Patent Application
20230349909
The present invention relates to the field of cancer immunotherapy. More specifically, the present invention relates to the prediction of responsiveness to cancer immunotherapy of a subject based on the T-cell composition of the subject, and a therapeutic method using cancer immunotherapy based on the prediction. In another aspect, the present invention provides a method of improving or maintaining responsiveness to cancer immunotherapy of a subject.
Cancer immunotherapy has drawn attention in recent years as having fewer side effects and exhibiting a greater effect compared to conventional anticancer therapies targeting the metabolism of cancer cells or the like (alkylating agents, platinum formulations, antimetabolites, topoisomerase inhibitors, microtubule polymerization inhibitors, microtubule depolymerization inhibitors, and the like). Among cancer immunotherapies, anti-PD-1 immune checkpoint inhibition has drawn particularly significant interest.
An anti-PD-1 antibody nivolumab is superior to docetaxel, which was conventionally the standard therapy as a secondary therapy of non-small cell lung cancer, by a large margin in all survival periods, thus becoming the standard therapy with a recommendation level of A in the Lung Cancer Society Guidelines (Brahmer J, et al. N Engl J Med 2015; 373: 123-135). Pembrolizumab, which is also an anti-PD-1 antibody, is superior to cytotoxic anticancer agents, which were conventionally the standard therapy in primary therapy, in all survival periods (note: in patients with expression of PD-L1 on tumor cells at 50% or greater). It has been decided that this will be the standard therapy for non-small cell lung cancer in the future.
The effect of anti-PD-1 antibodies is not limited to lung cancer. The effect is about to be proven in renal cancer, head and neck cancer, gastrointestinal cancer, gynecological cancer, malignant lymphoma, and breast cancer. Renal cancer is covered under insurance in Japan. Next year, head and neck cancer, gastrointestinal cancer, and malignant lymphoma are expected to be covered.
While anti-PD-1 antibodies appear to have achieved significant clinical success, anti-PD-1 antibodies in fact have significant problems. “Ineffective group”, whose condition worsens within three months in almost all anti-PD-1 antibody clinical trials, is found from data for progression free survival (PFS). Meanwhile, in groups for which anti-PD-1 antibodies were effective for 1 year or longer, exacerbation in conditions was hardly observed thereafter, thus revealing that a state close to being healed is attained. This suggests the presence of three different subgroups, i.e., “ineffective group”, “highly effective group” and “intermediate group” in terms of clinical effects, but a biomarker for the prediction thereof is not known. Administration of anti-PD-1 antibodies, which are expected to be the standard therapy in almost all cancer and tumor, to ineffective groups accounting for about 40%, which would be not only a medical problem, but also a problem for medical economics.
The present invention provides a method of using the composition of CD4+ T-cells of a subject as an indicator for predicting a response to cancer immunotherapy of the subject. The present invention also provides a method of using the composition of dendritic cells and/or CD8+ T-cells of a subject as an indicator for predicting a response of the subject to immunotherapy. The present invention is partially based on the inventors discovering that the responsiveness to cancer immunotherapy is associated with the composition of T-cells and/or dendritic cells in a subject, and the responsiveness can be used as a biomarker. The biomarker of the present invention has a much higher level of sensitivity and specificity than conventionally studied biomarkers.
The inventors have discovered that the three groups of therapeutic effects to cancer immunotherapy (e.g., anti-PD-1 therapy or anti-PD-L1 therapy), i.e., progressive disease (PD), stable disease (SD), and response (complete response (CR)+partial response (PR)), each exhibits different immunological conditions. Some of the embodiments of the present invention provide a method of predicting a response to cancer immunotherapy as either progressive disease (PD), stable disease (SD), or response (complete response (CR)+partial response (PR)) when cancer immunotherapy is applied to a subject. In the present invention, it should be noted that a population of subjects which includes complete response group (CR) with a partial response group (PR), or a population of subjects which includes a complete response group (CR) without a partial response group (PR), can be identified to be the same as a partial response group (PR).
One embodiment of the present invention is a method of using a relative amount of a CD4+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject. Examples of CD4+ T-cell subpopulations correlated with a dendritic cell stimulation in an anti-tumor immune response include, but are not limited to, CD4+ T-cell subpopulations with decreased expression of a homing molecule to a secondary lymphoid organ, CD4+ T-cell subpopulations primed by an effector T-cell, CD4+ T-cell subpopulations and regulatory T-cell subpopulations primed by antigen recognition.
The relative amount of a CD4+ T-cell subpopulation is selected from the group consisting of:
but is not limited thereto. The present invention provides, for example, a method of using a ratio of CD62Llow T-cells in CD4+ T-cells of a subject as an indicator for predicting a response to cancer immunotherapy of a subject. In one embodiment, the method comprises determining the ratio of CD62Llow T-cells in CD4+ T-cells in a sample derived from a subject. The ratio being higher than a threshold value (ineffective group threshold value) can indicate that the subject is not a part of an ineffective group with respect to the cancer immunotherapy.
Another embodiment of the present invention is a method of using a relative amount of a dendritic cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject. Examples of dendritic cell subpopulations correlated with a dendritic cell stimulation in an anti-tumor immune response include, but are not limited to, dendritic cell subpopulations that increase due to an increase in a cell subpopulation with decreased expression of a homing molecule in a CD4+ T-cell population and dendritic cell subpopulations that increase due to an increase in a CD4+ T-cell subpopulation primed by an effector T-cell in a CD4+ T-cell population. Examples of dendritic cell subpopulations include, but are not limited to, HLA-DR+ dendritic cell subpopulations, CD80+ dendritic cell subpopulations, CD86+ dendritic cell subpopulations, and PD-L1+ dendritic cell subpopulations. Examples of dendritic cells include, but are not limited to, myeloid dendritic cells (mDC, CD141+CD11c+ dendritic cells) and plasmacytoid dendritic cells (pDC, CD123+CD11c+ dendritic cells).
Another embodiment of the present invention is a method of using a relative amount of a CD8+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject. Examples of CD8+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response include, but are not limited to, CD8+ T-cell subpopulations that increase due to an increase in a cell subpopulation with decreased expression of a homing molecule in a CD4+ T-cell population, CD8+ T-cell subpopulations that increase due to an increase in a CD4+ T-cell subpopulation primed by an effector T-cell in a CD4+ T-cell population, CD8+ T-cell subpopulations that increase due to an increase in a CD4+ T-cell subpopulation primed by antigen recognition in a CD4+ T-cell population, CD8+ T-cell subpopulations that increase due to an increase in a HLA-DR+ dendritic cell subpopulation in a dendritic cell population, CD8+ T-cell subpopulations that increase due to an increase in a CD80+ T-cell subpopulation in a dendritic cell population, and CD8+ T-cell subpopulations that increase due to an increase in a PD-L1+ dendritic cell subpopulation in a dendritic cell population. Furthermore, examples of CD8+ T-cell subpopulations correlated with a dendritic cell stimulation in an anti-tumor immune response include, but are not limited to, CD62LlowCD8+ T-cell subpopulation, CD137+CD8+ T-cell subpopulation, and CD28+CD62LlowCD8+ T-cell subpopulation.
One embodiment of the present invention is a method of using an amount selected from:
In the present invention, (X) can be, for example, a value selected from the group consisting of:
For example, the method of the present invention can calculate variables (X, Y), with the amount of a regulatory T-cell subpopulation or the amount of a CD4+ T-cell subpopulation correlated with a regulatory T-cell as (Y). The method of the present invention can also calculate variables (X, Y), a value selected from the group consisting of:
The method of the present invention can use, for example, a comparison of a relative value of X to Y with a threshold value (ineffective group threshold value) comprising measuring the amount of CD4+CD62Llow T-cells (X) and measuring the amount of CD4+Foxp3+CD25+ T-cells (Y) as indicators for predicting that the subject is not a part of an ineffective group with respect to the cancer immunotherapy. The amount of a regulatory T-cell subpopulation or the amount or ratio of a CD4+ T-cell subpopulation correlated with a regulatory T-cell can be used as (Y). In particular, examination of the ratio of CD62Llow CD4+ T-cells/regulatory T-cells as a biomarker have not been reported up to this point, but the inventors have discovered that this ratio is very useful as a biomarker for predicting responsiveness to cancer immunotherapy.
The method of the present invention can use, for example, a comparison of a relative value of X to Y with a threshold value (ineffective group threshold value) comprising measuring the amount of CD80+ dendritic cells (X) and measuring the amount of a CD28+CD62LlowCD8+ T-cell (Y) as an indicator for predicting that the subject is not a part of an ineffective group with respect to the cancer immunotherapy.
Since the inventors have discovered that multiple indicators independently exhibit correlation with responsiveness, multiple indicators can be combined for use as an indicator for responsiveness. When two or more indicators are combined as an indicator for responsiveness, an indicator represented by a formula using any number of variables can be used. Examples of an indicator of responsiveness include, but are not limited to the following when multiple indicators (X1, X2, X3, . . . Xn) are used:
F=a
1
X
1
b1
+a
2
X
2
b2
+a
3
X
3
b3
. . . +a
n
X
n
bn
F=X
1
c1
*X
2
c2
*X
3
c3
. . . * X
n
cn
wherein each of a, b, and c is any real number. Responsiveness can be predicted from the difference derived from comparing a value calculated by such a formula (indicator) with a threshold value. Multivariate analysis (e.g., estimation by logistic regression) using discriminant analysis on the novel indicator discovered by the inventors can determine each coefficient for use as an indicator of responsiveness to cancer immunotherapy of a subject.
Typically, responsiveness can be predicted by formula F(X, Y) using two indicators (X, Y) disclosed herein as variables. In a specific embodiment, the formula is a relative value of X to Y.
Any function (F(X, Y)) of X and Y can be used as the relative value of X to Y. In particular, when X is considered to positively correlated with responsiveness and Y is negatively correlated with responsiveness, any function (F(X, Y)) of X and Y, which monotonically increases with respect to X and monotonically decreases with respect to Y, can be used, but is not limited thereto. A formula indicating responsiveness with two or more variables representing responsiveness can be found by regression by calculating the contribution of each variable to responsiveness.
Examples of formula F(X, Y) indicating responsiveness include, but are not limited to, the following:
F=aX
r
+bY
s
F=X
r
*Y
s
wherein a, b, r, and s are any real number.
For simplicity of the formula, an integer can be used as r and s. In some embodiments, examples of relative values of X to Y include, but are not limited to, Xn/Ym (n and m are any real number such as any integer) such as X/Y and X2/Y. When factors X and Y each indicates responsiveness to therapy from different mechanisms, such a combination of indicators can make prediction of responsiveness more accurate. The investigation by the inventors demonstrated that responsiveness to cancer immunotherapy of a subject can be predicted more accurately using a formula with r and s in the range of −5 to 5.
Another aspect of the present invention provides a method of further predicting a subject who is a part of a response group (complete response (CR)+partial response (PR)) from among subjects who have been shown to be not a part of an ineffective group by using the composition of a CD4+ T-cells of the subjects as an indicator of responsiveness to cancer immunotherapy.
One embodiment of the present invention is a method of using a ratio of a Foxp3+CD25+ T-cells in CD4+ T-cells, ICOS+CD62LlowCD4+ T cells in CD62LlowCD4+ T-cells, LAG-3+CD62LlowCD4+ T-cell subpopulation in CD62LlowCD4+ T-cells, or PD-1+CD62LlowCD4+ T-cell subpopulation in CD62LlowCD4+ T-cells in a subject, who is shown not to be a part of an ineffective group, as an indicator of a response to cancer immunotherapy of the subject. The ratio of Foxp3+CD25+ T-cells in CD4+ T-cells, the ratio of an ICOS+CD62LlowCD4+ T-cell subpopulation in CD62LlowCD4+ T-cells, the ratio of LAG-3+CD62LlowCD4+ T-cell subpopulation in CD62LlowCD4+ T-cells, or the ratio of PD-1+CD62LlowCD4+ T-cell subpopulation in CD62LlowCD4+ T-cells higher than an ineffective group threshold value can indicate that the subject is not a part of a response group. To determine whether a subject is a part of a response group, it is necessary to determine that the subject is not a part of an ineffective group. Such a determination of whether a subject is a part of an ineffective group can be achieved by a method disclosed herein.
Another embodiment of the present invention provides a method of identifying a response group (PR) and a stable group (SD) in a population of subjects determined not to be a part of an ineffective group using the aforementioned (X, Y). In a method of identifying a response group (PR) and stable group (SD), variables (Z, W) can be calculated, with an amount of an ICOS+CD62LlowCD4+ T-cell subpopulation as (Z) and a value selected from the group consisting of:
as (W) to predict whether a subject is a part of the response group (PR) or stable group (SD). Typically, a method of identifying a response group (PR) and a stable group (SD) can determine the response group (PR) and the stable group (SD) using the amount of an ICOS+CD62LlowCD4+ T-cell subpopulation as (Z), the amount of a CD4+Foxp3+CD25+ T-cell subpopulation as (W), and the value of W5*Z as an indicator.
A threshold value can be determined by considering sensitivity and specificity. Sensitivity and specificity can be sensitivity and specificity for the detection of an ineffective group, detection of a response group, or detection of a stable group. In one embodiment, a threshold value at which sensitivity and specificity are both 100% can be set for the biomarker of the present invention. For this reason, an ineffective case can be selected very accurately, so that this is technically superior in a significant manner. When two or more indicators disclosed as a biomarker of the present invention are used, threshold values can be determined for each indicator and used and distinguished as a first threshold value, second threshold value, third threshold value, and fourth threshold value as needed.
A threshold value can be determined so that sensitivity for the detection of an ineffective group, detection of a response group, or detection of a stable group exceeds about 90%. In another embodiment, a threshold value can be determined so that the sensitivity for the detection of an ineffective group, detection of a response group, or detection of a stable group is about 100%. In still another embodiment, a threshold value can be determined so that specificity for the detection of an ineffective group, detection of a response group, or detection of a stable group exceeds about 90%. In still another embodiment, a threshold value can be determined so that specificity for the detection of an ineffective group, detection of a response group, or detection of a stable group is about 100%.
In one embodiment, the composition of T-cells of a subject is a composition of T-cells in a sample obtained from the subject. Preferably, a sample is a peripheral blood sample. The biomarker provided in the present invention can be measured using a peripheral blood sample, so that the biomarker has significant superiority in clinical applications, e.g., non-invasive, low-cost, and implementable over time.
In one embodiment, cancer immunotherapy comprises administration of an immune checkpoint inhibitor. In particular, the biomarker of the present invention can accurately predict a response to such cancer immunotherapy of a subject.
In one embodiment, an immune checkpoint inhibitor comprises a PD-1 inhibitor or a PD-L1 inhibitor. Examples of PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies that inhibit an interaction between PD-1 and PD-L1 (e.g., binding) such as anti-PD-1 antibodies nivolumab and pembrolizumab. Examples of PD-L1 inhibitors include, but are not limited to, anti-PD-L1 antibodies that inhibit an interaction between PD-1 and PD-L1 (e.g., binding) such as anti-PD-L1 antibodies durvalumab, atezolizumab, and avelumab.
Still another aspect of the present invention provides a method of predicting a response to cancer immunotherapy of a subject using the composition of T-cells of the subject to treat the subject with cancer. Alternatively, a method of treating cancer in a subject with a specific composition of T-cells or a composition therefor is provided. Cancer immunotherapy, especially immune checkpoint inhibition therapy is known to have a wide difference in responsiveness for each subject. Administration of cancer immunotherapy by selecting a subject using the biomarker of the present invention can significantly increase the probability of achieving a therapeutic effect such as tumor regression.
One embodiment of the present invention provides a method of treating a subject with cancer, comprising:
Another embodiment of the present invention provides a method of treating a subject with cancer, comprising applying a cancer immunotherapy to a subject if the subject is determined to be not a part of an ineffective group by determining a relative amount selected from the group consisting of:
In this method, the relative amount is selected from the group consisting of:
Preferably, the relative amount is selected from the group consisting of:
Another embodiment of the present invention provides a method of treating a subject with cancer, comprising: determining a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells in a sample derived from the subject; determining that the subject is not a part of an ineffective group with respect to a response to cancer immunotherapy if the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells is lower than a threshold value (ineffective group threshold value); and applying the cancer immunotherapy to the subject if the subject is determined to be not a part of an ineffective group. Another embodiment of the present invention provides a method of treating a subject with cancer, comprising applying the cancer immunotherapy to the subject who is determined not a part of an ineffective group with respect to a response to cancer immunotherapy by: determining a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells in a sample derived from the subject; and determining that the subject is not a part of an ineffective group with respect to a response to cancer immunotherapy if the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells is lower than a threshold value (ineffective group threshold value).
Another embodiment of the present invention provides a method of treating a subject with cancer, comprising:
Another embodiment of the present invention provides a method of treating a subject with cancer, comprising applying the cancer immunotherapy to the subject who is determined not a part of an ineffective group with respect to a response to cancer immunotherapy by:
For example, the aforementioned amounts (X) and (Y) are selected from the group consisting of:
For example, the method of the present invention can use a value selected from the group consisting of:
For example, the method of the present invention can calculate variables (X, Y), with an amount of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells as (Y). The method of the present invention can also calculate variables (X, Y), with a value selected from the group consisting of:
Another embodiment of the present invention provides a method of treating a subject with cancer, comprising: determining amounts (X, Y) selected from the group consisting of:
For example, method of the present invention can use the value selected from the group consisting of:
For example, the method of the present invention can calculate variables (X, Y), with an amount of a regulatory T-cell subpopulation or a CD4+ T-cell subpopulation correlated with regulatory T-cells as (Y). The method of the present invention can also calculate variables (X, Y), with a value selected from the group consisting of:
Another aspect of the present invention provides a method of identifying a response group (PR) and a stable group (SD) in a population of subjects determined to be not a part of an ineffective group using the aforementioned (X, Y). A method of identifying a response group (PR) and a stable group (SD) can calculate variables (Z, W), with an amount of an ICOS+CD62LlowCD4+ T-cell subpopulation as (Z) and a value selected from the group consisting of:
Still another aspect of the present invention provides a kit for predicting a response to cancer immunotherapy of a subject comprising an agent for detecting one or more cell surface markers selected from CD4, CD25, CD62L, Foxp3, and the like, such as a combination of markers selected from the group consisting of:
One embodiment of the present invention is a kit comprising an agent for detecting a cell surface marker for predicting a response to cancer immunotherapy of a subject. The inventors discovered that these cell surface markers expressed by a T-cell of a subject are related to responsiveness to cancer immunotherapy of a subject. It is understood that a kit comprising an agent for detecting such cell surface markers are useful in predicting responsiveness to cancer immunotherapy in view of the above. A kit preferably comprises an agent for detecting CD4 and CD62L. A kit more preferably comprises an agent for detecting CD4, CD25, CD62L, and Foxp3. In one embodiment, a detection agent is an antibody. Preferably, an antibody facilitates the detection of a suitably labeled marker.
Another aspect of the present invention is a composition for treating cancer in a subject, comprising an immune checkpoint inhibitor.
One embodiment of the present invention is a composition for treating cancer in a subject, comprising an immune checkpoint inhibitor, characterized in that the subject has a relative amount selected from the group consisting of:
For example, the relative amount is typically selected from the group consisting of:
Still another embodiment of the present invention is a composition for treating cancer in a subject, comprising an immune checkpoint inhibitor, characterized in that the subject is a subject selected by comparison of a threshold value (ineffective group threshold value) and a relative value of X to Y with amounts (X, Y) selected from the group consisting of:
For example, the method of the present invention can use a value selected from the group consisting of:
For example, method of the present invention can calculate variables (X, Y), with the amount of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells as (Y). The method of the present invention can also calculate variables (X, Y), with a value selected from the group consisting of:
For example, the method of the present invention can use a comparison of a relative value of X to Y with a threshold value (ineffective group threshold value), comprising measuring the amount of CD4+CD62Llow T-cells (X) and measuring the amount of CD4+Foxp3+CD25+ T-cells (Y) as an indicator for predicting that the subject is not a part of an ineffective group with respect to the cancer immunotherapy. The amount or ratio of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells can be used as (Y).
Still another embodiment of the present invention is a composition for treating cancer in a subject, comprising an immune checkpoint inhibitor, characterized in that the subject is a subject selected by comparison of a threshold value (ineffective group threshold value) with a relative value of amounts (X, Y) selected from the group consisting of:
For example, the target of administration of the composition of the present invention can be a subject characterized by variables (X, Y), with a value selected from the group consisting of:
For example, for the composition of the present invention, variables (X, Y) can be calculated, with an amount of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells as (Y). The method of the present invention can also calculate variables (X, Y), with a value selected from the group consisting of:
For example, a subject predicted to be not a part of an ineffective group with respect to cancer immunotherapy can be targeted as the target of administrating the composition of the present invention by comparing a relative value of X to Y with a threshold value (ineffective group threshold value) from the amount of CD4+CD62Llow T-cells (X) and the amount of CD4+Foxp3+CD25+ T-cells (Y). The amount or ratio of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells can be used as (Y). The composition of the present invention can be used in combination with any other agents.
In one embodiment, a composition comprises a PD-1 inhibitor. Examples of PD-1 inhibitors include anti-PD-1 antibodies that inhibit a binding between PD-L1 and PD-1, such as nivolumab or pembrolizumab. In another embodiment, the composition comprises a PD-L1 inhibitor. Examples of PD-L1 inhibitors include anti-PD-L1 antibodies that inhibit a binding between PD-L1 and PD-1, such as durvalumab, atezolizumab, and avelumab. Compositions comprising such immune checkpoint inhibitors are understood as attaining a therapeutic effect at an especially high probability when administered to a subject selected using the biomarker of the present invention.
Another aspect of the present invention provides a method of improving or maintaining responsiveness to cancer immunotherapy of a subject. It was discovered that cells selected from the group consisting of:
CD62LlowCD4+ T-cells;
are important to the response of a subject to cancer immunotherapy. It is understood that use of such T-cells can improve or maintain the responsiveness to cancer immunotherapy of a subject. One embodiment of the present invention is a composition comprising a CD62LlowCD4+ T-cell. A CD62LlowCD4+ T-cell or a composition comprising the same is useful for treating or preventing cancer and can be used in combination with cancer immunotherapy. In a still another embodiment, the composition may comprise a CD62LlowCD8+ T-cell in addition to CD62LlowCD4+ T-cell or the like.
One embodiment of the present invention provides a method of making cancer immunotherapy effective in a subject for whom the cancer immunotherapy is predicted to be ineffective by using a cell selected from the group consisting of:
or a composition therefor. Another embodiment of the present invention provides a method for sustaining an effect of cancer immunotherapy by using, for example, CD62LlowCD4+ T-cells or a composition thereof. The results of the inventors' study, which elucidated that CD62LlowCD4+ T-cells of peripheral blood play the role of an accelerator for an anti-tumor immune response in cancer immunotherapy (especially anti-PD-1 antibody therapy and/or anti-PD-L1 antibody therapy), is a novel finding, which provides a new and revolutionary approach to cancer therapy. The inventors have also discovered that a cell selected from the group consisting of:
Some embodiments of the present invention provide a method of refining or purifying CD62LlowCD4+ T-cells or a method of manufacturing a composition comprising CD62LlowCD4+ T-cells. In some embodiments, CD62LlowCD4+ T cells are isolated from a human derived sample. Some embodiments provide a method of preparing CD62LlowCD4+ T cells that have been isolated from a subject for infusion into the subject and a method of infusing such cells into the subject. One embodiment of the present invention is a composition comprising a CD62LlowCD4+ T-cell, which is from a subject to whom the composition is administered. A still another aspect of the present invention provides a method of refining or purifying CD62LlowCD8+ T-cells or a method of manufacturing a composition comprising CD62LlowCD8+ T-cells. Using the same principle, the present invention similarly prepares a cell selected from the group consisting of:
A method of manufacturing a composition comprising CD62LlowCD4+ T-cells can comprise purifying CD62LlowCD4+ T-cells from a T-cell population derived from a human. The purifying may comprise removing a CD62L high expression cell from the T-cell population (negative selection) Purification of CD62LlowCD4+ T-cells by negative selection using an antibody and/or magnetic beads and/or affinity column, or the like is preferable because impurities such as an antibody or magnetic beads do not remain on a cell to be used. The present invention also provides a method of manufacturing a composition comprising CD62LlowCD8+ T-cells.
One embodiment of the present invention is a kit comprising a substance, which specifically binds to CD62L, for purifying CD62LlowCD4+ T-cells. Examples of a substance which specifically binds to CD62L include, but are not limited to, antibodies that are specific to CD62L.
(Biomarkers of the Present Invention)
The biomarker of the present invention is understood to evaluate the overall balance of anti-tumor immune responses, including CD4+ T-cells, dendritic cells, and/or CD8+ T-cells to evaluate the overall tumor immunity itself. For this reason, the method of the present invention can be considered effective against a wide range of cancers and tumors. The present invention evaluates the overall anti-tumor immune responses, so that the present invention is also expected to be effective for not only an immune checkpoint inhibitor against PD-1/PD-L1, but also anticancer therapy which acts on other immune checkpoints.
The present invention can also use a marker, which is indicative of an effector T-cell, such as CCR7−, instead of or in addition to CD62Llow. Alternatively, CD45RA− and/or CD45RO+ can be used. For example, the ratio of a CD45RA−CD4+ T-cell subpopulation in CD4+ T-cells and/or the ratio of CD45RO+CD4+ T-cell subpopulation in CD4+ T-cells can also be used. It was revealed that expression of LAG3 and ICOS can also be used (added or substituted) in a similar manner to CD62Llow. It was similarly discovered that CCR4 expression can also be used (added or substituted) in a similar manner to CD62Llow.
Instead of (or in addition to) using CD4+ T-cells (CD62LlowCD4+ T-cells) which were used in the Examples, the number/ratio of cells expressing HLA-DR and/or CD80 and/or CD86 in a myeloid dendritic cell (mDC) and/or plasmacytoid dendritic cell (pDC) population can also be used as an indicator. PD-L1 on dendritic cells is also understood to be available as the marker of the present invention.
Further, instead (or in addition to) of using CD4+ T-cells (CD62LlowCD4+ T-cells) which were used in the Examples, the number/ratio of cells expressing 4-1BB in CD8+ T-cells can also be used as an indicator.
(Mechanism of the Present Invention)
Although not wishing to be bound by any theory, the anti-tumor immune response phenomenon at a local tumor proposed by the inventors is schematically shown in
The composition of T-cells is considered important in anti-tumor immune responses. For example, a stimulation of a dendritic cell by a CD62LlowCD4+ T-cell is important. If there are insufficient CD62LlowCD4+ T-cells (e.g., the balance of effector T-cells and naive T-cells leans towards naive T-cells), dendritic cells cannot be sufficiently stimulated even with administration of an immune checkpoint inhibitor. As a result, an anti-tumor immune response cannot be sufficient. For this reason, the ratio of CD62LlowCD4+ T-cells in CD4+ T-cells is an indicator for predicting an anti-tumor effect due to an immune checkpoint inhibitor. As with CD62L, the ratio of CD45RA− CCR7− T-cells in CD4+ T-cells also indicates the balance of effector T-cells and naive T-cells, so that such a ratio can be used as an indicator of the present invention.
Dendritic cells are stimulated by CD4+ T-cells via HLA-DR. Thus, with a decrease in the ratio of HLA-DR+ cells in dendritic cells, the dendritic cells cannot be sufficiently stimulated even with administration of an immune checkpoint inhibitor. As a result, anti-tumor immune responses cannot be sufficient. For this reason, the ratio of HLA-DR+ cells in dendritic cells can also be an indicator for predicting an anti-tumor effect due to an immune checkpoint inhibitor.
Dendritic cells which have been stimulated by CD4+ T-cells stimulate CD8+ T-cells, and stimulated CD8+ T-cells ultimately exert anti-tumor activity. CD8+ T-cells are stimulated by dendritic cells via CD80/CD86 expressed on dendritic cells and CD137 on CD8+ T-cells. Thus, both the ratio of CD80+ cells in dendritic cells and the ratio of CD137+ cells in CD8+ T-cells can be an indicator for predicting an anti-tumor effect due to an immune checkpoint inhibitor.
In addition to the biomarkers revealed from the mechanism disclosed above, LAG-3, ICOS, and CCR4 in CD4+ T-cells were also found to be an indicator for predicting an anti-tumor effect due to an immune checkpoint inhibitor as disclosed in the Examples.
For example, the present invention provides the following items.
(Item 1)
A method of using a relative amount of a CD4+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject, comprising determining the relative amount of the CD4+ T-cell subpopulation in a sample derived from the subject, the relative amount higher than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item 2)
The method of item 1, wherein the relative amount of the CD4+ T-cell subpopulation is selected from the group consisting of:
(Item 3)
A method of using a ratio of CD62Llow T-cells in CD4+ T-cells of a subject as an indicator for predicting a response to cancer immunotherapy of the subject, comprising determining the ratio of CD62Llow T-cells in the CD4+ T-cells in a sample derived from the subject, the ratio higher than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item 4)
A method of using a relative amount of a dendritic cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject, comprising determining a ratio of the dendritic cell subpopulation in dendritic cells in a sample derived from the subject, the ratio higher than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item 5)
The method of item 4, wherein the dendritic cell subpopulation is selected from the group consisting of: an HLA-DR+ dendritic cell subpopulation; a CD80 dendritic cell subpopulation; a CD86+ dendritic cell subpopulation; and PD-L1+ dendritic cell subpopulation.
(Item 6)
A method of using a relative amount of a CD8+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response as an indicator for predicting a response to cancer immunotherapy of a subject, comprising determining a ratio of the CD8+ T-cell subpopulation in CD8+ T-cells in a sample derived from the subject, the ratio higher than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item 7)
The method of item 6, wherein the CD8+ T-cell subpopulation is a CD62LlowCD8+ T-cell subpopulation, a CD137+CD8+ T-cell subpopulation, or a CD28+CD62LlowCD8+ T-cell subpopulation.
(Item 8)
A method of using a relative value of amounts (X, Y) selected from the group consisting of:
(Item 9)
The method of item 8, wherein the amounts (X) and (Y) are respectively selected from the group consisting of:
(Item 10)
The method of item 8 or 9, wherein the relative value is X/Y.
(Item 11)
The method of item 8 or 9, wherein the relative value is X2/Y.
(Item 12)
The method of any one of items 1 to 11, further using
(Item 13)
The method of any one of items 1 to 12, wherein the ineffective group threshold value is determined by considering sensitivity and specificity for the detection of an ineffective group.
(Item 14)
The method of any one of items 1 to 13, wherein the ineffective group threshold value is determined so that sensitivity for the detection of an ineffective group exceeds about 90%.
(Item 15)
The method of any one of items 1 to 13, wherein the ineffective group threshold value is determined so that specificity for the detection of an ineffective group exceeds about 90%.
(Item 16)
The method of any one of items 1 to 15, wherein the sample is a peripheral blood sample.
(Item 17)
The method of any one of items 1 to 16, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
(Item 18)
The method of item 17, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item 19)
The method of item 18, wherein the PD-1 inhibitor is an anti-PD-1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item 20)
The method of item 18, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item 21)
The method of item 18, wherein the PD-1 inhibitor or PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item 22)
A method of treating a subject with cancer, comprising:
(Item 23)
The method of item 22, wherein the relative amount is selected from the group consisting of:
(Item 24)
A method of treating a subject with cancer, comprising:
(Item 25)
The method of item 24, wherein the amounts (X) and (Y) are respectively selected from the group consisting of:
(Item 26)
A method of treating a subject with cancer, comprising:
determining amounts (X, Y) selected from the group consisting of:
(Item 27)
A kit for predicting a response to cancer immunotherapy of a subject, comprising an agent for detecting a combination of markers selected from the group consisting of:
(Item 28)
The kit of item 27, comprising an agent for detecting CD4 and CD62L.
(Item 29)
The kit of item 27, comprising an agent for detecting CD4, CD25, CD62L, and Foxp3.
(Item 30)
The kit of any one of items 27 to 29, wherein the detection agent is an antibody.
(Item 31)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject has a relative amount selected from the group consisting of:
(Item 32)
The composition of item 31, wherein the relative amount is selected from the group consisting of:
(Item 33)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject is a subject selected by comparison of an ineffective group threshold value with a relative value of amounts (X, Y) selected from the group consisting of:
(Item 34)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject is a subject selected by comparison of an ineffective group threshold value with a relative value of amounts (X, Y) selected from the group consisting of:
(Item 35)
The composition of any one of items 31 to 34, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item 36)
The composition of item 35, wherein the PD-1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-L1 and PD-1.
(Item 37)
The composition of item 35, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item 38)
The composition of item 35, wherein the PD-1 inhibitor of PD-L1 inhibitor is nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item 39)
A composition for treating or preventing cancer, comprising a cell selected from the group consisting of:
(Item 40)
The composition of item 39 for concomitant use with cancer immunotherapy.
(Item 41)
The composition of item 39, characterized in that the composition is administered in combination with an immune checkpoint inhibitor.
(Item 42)
The composition of item 41, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item 43)
The composition of item 42, wherein the PD-1 inhibitor is an anti-PD-1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item 44)
The composition of item 42, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item 45)
The composition of item 42, wherein the PD-1 inhibitor or PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item 46)
The composition of any one of items 39 to 45, further comprising a CD62LlowCD8+ T-cell.
(Item 47)
The composition of any one of items 39 to 46 for making cancer immunotherapy effective in a subject to whom cancer immunotherapy is predicted to be ineffective.
(Item 48)
The composition of any one of items 39 to 47 for sustaining an effect of cancer immunotherapy.
(Item 49)
The composition of any one of items 39 to 48, wherein the CD62LlowCD4+ T-cell is from a subject to whom the composition is administered.
(Item 50)
A method of manufacturing a composition for treating or preventing cancer comprising CD62LlowCD4+ T-cells, comprising purifying CD62LlowCD4+ T-cells from a T-cell population derived from a human.
(Item 51)
The method of item 50, wherein the purifying comprises removing a CD62L high expression cell from a T-cell population.
(Item 52)
A kit comprising a substance, which specifically binds to CD62L, for purifying CD62LlowCD4+ T-cells.
(Item A1)
A method of using a ratio of CD62Llow T-cells in CD4+ T-cells of a subject as an indicator for predicting a response to cancer immunotherapy of the subject, comprising determining the ratio of CD62Llow T-cells in CD4+ T-cells in a sample derived from the subject, the ratio higher than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item A2)
A method of using a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells of a subject as an indicator for predicting a response to cancer immunotherapy of the subject, comprising determining the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells in a sample derived from the subject, the ratio lower than an ineffective group threshold value indicating that the subject is not a part of an ineffective group to the cancer immunotherapy.
(Item A3)
A method of using a relative value of an amount of CD4+CD62Llow T-cells of a subject (X) to an amount of CD4+Foxp3+CD25+ T-cells (Y) as an indicator for predicting a response to cancer immunotherapy of the subject, the method comprising:
(Item A4)
The method of item A3, wherein the relative value is X/Y.
(Item A5)
The method of item A3, wherein the relative value is X2/Y.
(Item A6)
The method of any one of items A1 to A5, further using a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells in a subject who is shown not to be a part of the ineffective group as an indicator of a response to cancer immunotherapy of the subject, wherein the ratio of the Foxp3+CD25+CD4+ T-cells in the CD4+ T-cells higher than a response group threshold value indicates that the subject is a part of a response group.
(Item A7)
The method of any one of items A1 to A6, wherein the ineffective group threshold value is determined by considering sensitivity and specificity for the detection of an ineffective group.
(Item A8)
The method of any one of items A1 to A7, wherein the ineffective group threshold value is determined so that sensitivity for the detection of an ineffective group exceeds about 90%.
(Item A9)
The method of any one of items A1 to A8, wherein the ineffective group threshold value is determined so that specificity for the detection of an ineffective group exceeds about 90%.
(Item A10)
The method of any one of items A1 to A9, wherein the sample is a peripheral blood sample.
(Item A1 l)
The method of any one of items A1 to A10, wherein the cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
(Item A12)
The method of item A11, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item A13)
The method of item A12, wherein the PD-1 inhibitor is an anti-PD-1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A14)
The method of item A12, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A15)
The method of item A12, wherein the PD-1 inhibitor or PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item A16)
A method of treating a subject with cancer, comprising:
(Item A17)
A method of treating a subject with cancer, comprising:
(Item A18)
A method of treating a subject with cancer, comprising:
(Item A19)
A method of treating a subject with cancer, comprising:
(Item A20)
A kit for predicting a response to cancer immunotherapy of a subject, comprising an agent for detecting one or more cell surface markers selected from CD4, CD25, CD62L, and Foxp3.
(Item A21)
The kit of item A20, comprising an agent for detecting CD4 and CD62L.
(Item A22)
The kit of item A20 or A21, comprising an agent for detecting CD4, CD25, CD62L, and Foxp3.
(Item A23)
The kit of any one of items A20 to 22, wherein the detection agent is an antibody.
(Item A24)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject has a ratio of CD62Llow T-cells in CD4+ cells in a sample derived from the subject, wherein the ratio is equal to or greater than an ineffective group threshold value.
(Item A25)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject has a ratio of Foxp3+CD25+ T-cells in CD4+ cells in a sample derived from the subject, wherein the ratio is equal to or less than an ineffective group threshold value.
(Item A26)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject is a subject selected by comparison of an ineffective group threshold value with a relative value of an amount of CD4+CD62LlowT-cells (X) in a sample derived from the subject to an amount of CD4+Foxp3+CD25+ T-cells (Y) in a sample derived from the subject.
(Item A27)
A composition comprising an immune checkpoint inhibitor for treating cancer in a subject, characterized in that the subject is a subject selected by comparison of an ineffective group threshold value with a relative value of an amount of CD4+CD62LlowT-cells (X) in a sample derived from the subject to an amount of CD4+Foxp3+CD25+ T-cells (Y), and in a sample derived from the subject, having a ratio of Foxp3+CD25+ T-cell subpopulation in CD4+ T-cells being equal to or higher than a response group threshold value.
(Item A28)
The composition of any one of items A24 to A27, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item A29)
The composition of item A28, wherein the PD-1 inhibitor is an anti-PD-1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A30)
The composition of item A28, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A31)
The composition of item A28, wherein the PD-1 inhibitor or PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item A32)
A composition for treating or preventing cancer, comprising a CD62LlowCD4+ T-cell.
(Item A33)
The composition of item A32 for concomitant use with cancer immunotherapy.
(Item A34)
The composition of item A32, characterized in that the composition is administered in combination with an immune checkpoint inhibitor.
(Item A35)
The composition of items A34, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor and a PD-L1 inhibitor.
(Item A36)
The composition of item A35, wherein the PD-1 inhibitor is an anti-PD-1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A37)
The composition of item A35, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.
(Item A38)
The composition of item A35, wherein the PD-1 inhibitor or PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab, or avelumab.
(Item A39)
The composition of any one of items A32 to A38, further comprising a CD62LlowCD8+ T-cell.
(Item A40)
The composition of any one of items A32 to A39 for making cancer immunotherapy effective in a subject for whom the cancer immunotherapy is predicted to be ineffective.
(Item A41)
The composition of any one of items A32 to A40 for sustaining an effect of cancer immunotherapy.
(Item A42)
The composition of any one of items A32 to A41, wherein the CD62LlowCD4+ T-cell is from a subject to whom the composition is administered.
(Item A43)
A method of manufacturing a composition for treating or preventing cancer comprising CD62LlowCD4+ T-cells, comprising purifying CD62Llow CD4+ T-cells from a T-cell population derived from a human.
(Item A44)
The method of item A43, wherein the purifying comprises removing a CD62L high expression cell from a T-cell population.
(Item A45)
A kit comprising a substance, which specifically binds to CD62L, for purifying CD62LlowCD4+ T-cells.
The present invention is disclosed hereinafter using exemplary Examples while referring to the appended drawings as needed. Throughout the entire specification, a singular expression should be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Further, the terms used herein should be understood to be used in the meaning that is commonly used in the art, unless specifically noted otherwise. Thus, unless defined otherwise, all terminologies and scientific technical terms that are used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of a contradiction, the present specification (including the definitions) takes precedence.
As used herein, “biomarker” refers to characteristics that can be objectively measured and evaluated as an indicator of a normal biological process, pathological process, or a pharmacological response to therapeutic intervention.
As used herein, “cancer” refers to malignant tumor, which is highly atypical, expands faster than normal cells, and can destructively infiltrate or metastasize surrounding tissue, or the presence thereof. In the present invention, cancer includes, but is not limited to, solid cancer and hematopoietic tumor.
As used herein, “cancer immunotherapy” refers to a method of treating cancer using a biological defense mechanism such as the immune mechanism of organisms.
As used herein, “anti-tumor immune response” refers to any immune response against tumor in a live organism.
As used herein, “dendritic cell stimulation in an anti-tumor immune response” refers to any phenomenon applying a stimulation to dendritic cells, which occurs in the process of an immune response against tumor in a live organism. Such stimulation can be a direct or indirect factor for inducing an anti-tumor immune response. Although not limited to the following, typically, a dendritic cell stimulation in an anti-tumor immune response is applied by CD4+ T-cells (e.g., effector T-cells), resulting in dendritic cells stimulating CD8+ T-cells, and the stimulated CD8+ T-cells exerting an anti-tumor effect.
As used herein, “correlation” refers to two matters having a statistically significant correlated relationship For example, “relative amount of B correlated with A” refers to the relative amount of B being statistically significantly affected (e.g., increase or decrease) when A occurs.
As used herein, “cell subpopulation” refers to a cell population constituting a part of an entire cell population.
As used herein, the term “relative amount” with regard to cells can be interchangeably used with “ratio”. Typically, the terms “relative amount” and “ratio” refer to the number of cells constituting a given cell subpopulation (e.g., CD62LlowCD4+ T-cells) with respect to the number of cells constituting a specific cell population (e.g., CD4+ T-cell population).
As used herein, “sensitivity” refers to the ratio of number of subjects having a given feature in selected targets to the total number of subjects with the given feature in a subject population when selecting a subject with the given feature from among a population of subjects, i.e., (number of subjects having a given feature in a selected target)/(total number of subjects with the given feature in a subject population).
As used herein, “specificity” refers to the ratio of the number of subjects with a given feature in selected targets to the total number of selected targets when selecting a subject with the given feature from among a population of subjects, i.e., (number of subjects with a given feature among selected targets)/(total number of selected targets).
As used herein, “ineffective group” refers to a group of subjects determined to be progressive (PD, Progressive disease) when a therapeutic effect upon undergoing cancer therapy is determined according to RECIST ver 1.1 at the early stage of up to about 9 weeks after start of the treatment. An ineffective group is also called a PD group, progressive group, or NR (Non-responder) which are interchangeably used herein.
As used herein, “partial response group” refers to a group of subjects determined to be partial response (PR, Partial response) when a therapeutic effect upon undergoing cancer therapy is determined according to RECIST ver 1.1. A partial response group is also called a PR group, which is interchangeably used herein.
As used herein, “stable group” refers to a group of subjects determined to be stable (SD, Stable disease) when a therapeutic effect upon undergoing cancer therapy is determined according to RECIST ver 1.1 at the early stage of up to about 9 weeks after start of the treatment. A “stable group” is also called an SD group or intermediate group, which are interchangeably used herein. Further, once the group turns into disease progression about 1 year after the disease control, this group is called IR (Intermediate Responder). Since most of this group is determined as SD about 9 weeks after the start of the treatment, “stable group” is also used interchangeably with IR (Intermediate Responder) group.
As used herein, “complete response group” refers to a group of subjects determined to be complete response (CR, Complete response) when a therapeutic effect upon undergoing cancer therapy is determined according to RECIST ver 1.1. A “complete response group” is also called a CR group, which is interchangeably used herein. The present invention detects a case where a population of subjects comprises a complete response group (CR) in addition to a partial response group (PR) and a case where a population of subjects comprises a complete response group (CR) without comprising a partial response group (PR) as the same as a partial response group (PR).
As used herein, “response group” is used when a “partial response group” and “complete response group” are collectively called. This is also called a “highly effective group”. In addition, a group where a long term disease state control lasted for more than 1 year after starting the treatment is called GR (Good responder). However, since most of this group is identified as “partial response group” or “complete response group” 9 weeks after starting treatment, “response group” can also be used interchangeably with GR (Good responder) group.
As used herein, “relative value” refers to a value obtained by calculating a certain value while using another value as a baseline of comparison.
As used herein, the term “detection agent” broadly refers to all agents that are capable of detecting a substance of interest (e.g., cell surface marker or the like).
As used herein, the “amount” of a certain cell subpopulation encompasses the absolute number of certain cells and relative amount of a ratio in a cell population.
As used herein, “threshold value” refers to a value that is determined for a certain variable value, where the value gives a certain meaning when the changing value is greater or less than the value. A threshold value is also called a cut-off value herein.
As used herein, “ineffective group threshold value” refers to a threshold value used for identifying an ineffective group and stable group+response group in a given population of subjects. An ineffective group threshold value is selected to achieve a predetermined sensitivity and specificity when selecting an ineffective group in a given population of subjects.
As used herein, “response group threshold value” refers to a threshold value used for identifying a stable group and a response group in a given population of subjects or in a given population of subjects from which an ineffective group is removed using an ineffective group threshold value. A response threshold value is selected to achieve a predetermined sensitivity and specificity when selecting a response group in a given population of subjects or in a given population of subjects from which an ineffective group is removed using an ineffective group threshold value.
The term “about”, when used to qualify a numerical value herein, is used to mean that the described value encompasses a range of values up to +10%.
As used herein, “flow cytometry” refers to a technique of measuring the number of cells, individual or other biological particles suspended in a liquid and individual physical/chemical/biological attributes.
(Cancer Immunotherapy)
Cancer immunotherapy is a method of treating cancer using a biological defense mechanism of an organism. Cancer immunotherapy can be largely divided into cancer immunotherapy from strengthening the immune function against cancer and cancer immunotherapy from inhibiting the immune evasion mechanism of cancer. Cancer immunotherapy further includes active immunotherapy for activating the immune function in the body and passive immunotherapy for returning immune cells with an immune function activated or expanded outside the body into the body. The biomarker of the present invention is understood to evaluate the overall balance of the CD4+ T-cell immunity to evaluate the overall tumor immunity itself, so that a therapeutic effect of all cancer immunotherapy can be broadly predicted.
Examples of cancer immunotherapy include non-specific immunopotentiators, cytokine therapy, cancer vaccine therapy, dendritic cell therapy, adoptive immunotherapy, non-specific lymphocyte therapy, cancer antigen specific T-cell therapy, antibody therapy, immune checkpoint inhibition therapy and the like. The Examples of the present specification demonstrate that the biomarker of the present invention accurately predicts a therapeutic effect of especially, although not limited to, immune checkpoint inhibition therapy.
PD-1 inhibitors are representative examples of immune checkpoint inhibitors. Examples of PD-1 inhibitors include, but are not limited to, anti-PD-1 antibody nivolumab (sold as Opdivo™) and pembrolizumab. In one preferred embodiment, nivolumab can be selected. Although not wishing to be bound by any therapy, one of the reasons that therapy using nivolumab is preferred is because the Examples demonstrate that the use of the biomarker of the present invention can clearly identify a responsive subject and a non-responsive subject, and especially because it is revealed that responsiveness and non-responsiveness can be clearly distinguished by a specific threshold value. Of course, it is understood that the biomarker of the present invention can be utilized for other PD-1 inhibitors to the same degree.
The present invention can also use PD-L1 inhibitors to the same extent as PD-1 inhibitors.
It is understood that anti-PD-1 antibodies achieve an anti-cancer effect by releasing the suppression of T-cell activation by a PD-1 signal. It is understood that anti-PD-L1 antibodies also achieve an anticancer effect by releasing the suppression of T-cell activation by a PD-1 signal. While the mechanism of PD-1 inhibiting a T-cell function is not fully elucidated, it is understood that an interaction between PD-1 (programmed death 1) and PD-L1 or PD-L2 recruits a tyrosine phosphatase, SUP-1 or 2, to the cytoplasmic domain of PD-1 to inactivate a T-cell receptor signaling protein ZAP70 to suppress activation of T-cells (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 understood to be caused by the recruitment of SUP-1 or 2 to a part called an ITSM motif which dephosphorylates proximal signaling kinase of a T-cell receptor in the vicinity. In other words, the memory of “being stimulated by an antigen” is erased from a T-cell that has been stimulated by an antigen.
PD-1 is expressed at a high level in killer T-cells and natural killer cells, which have infiltrated a cancer tissue. It is understood that an immune response mediated by a PD-1 signal from PD-1 is attenuated by PD-L1 on tumor. While the immune response mediated by a PD-1 signal is attenuated by PD-L1, an effect of enhancing an anti-tumor immune response is attained by inhibiting an interaction between PD-1 and PD-L1 and/or signaling induced by an interaction with an anti-PD-1 antibody.
A PD-L1 inhibitor (e.g., anti-PD-L1 antibodies avelumab, durvalumab, and atezolizumab) is another example of an immune checkpoint inhibitor.
PD-L1 inhibitors bind and inhibit the aforementioned PD-1 pathway to the PD-L1 side to inhibit an interaction between PD-1 and PD-L1 and/or signaling induced by an interaction to induce an anti-tumor immune response. Although not wishing to be bound by any therapy, subjects who are responsive or non-responsive to therapy that inhibits the PD-1 pathway (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) can be clearly identified by using the biomarker of the present invention in view of the results demonstrated in the Examples.
A CTLA-4 inhibitor (e.g., anti-CTLA-4 antibody ipilimumab or tremelimumab) is another example of an immune checkpoint inhibitor.
CTLA-4 inhibitors activate T-cells to induce an anti-tumor immune response. T-cells are activated by an interaction of CD28 on the surface with CD80 or CD86. However, it is understood that surface expressed CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) preferentially interacts with CD80 or CD86 with higher affinity than CD20 to suppress activation, even for T-cells that have been activated. CTLA-4 inhibitors induce an anti-tumor immune response by inhibiting CTLA-4 to prevent inhibition of an interaction between CD20 and CD80 or CD86.
In another embodiment, an immune checkpoint inhibitor may target an immune checkpoint protein 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 immuno-receptor with Ig and ITIM domain).
It is understood that the aforementioned immune checkpoints also suppress an immune response to autologous tissue, but immune checkpoints increase in T-cells when an antigen such as a virus is present in vivo for an extended period of time. It is understood that tumor tissue is also an antigen which is present in vivo for an extended period of time, so that an anti-tumor immune response is evaded by such immune checkpoints. The aforementioned immune checkpoint inhibitors invalidate such an evasion function to achieve an anti-tumor effect. Although not wishing to be bound by any therapy, it is understood that the biomarker of the present invention evaluates the balance of the overall anti-tumor immune responses of humans so that it can be used as an indicator for accurately predicting a therapeutic effect of such an immune checkpoint inhibitor.
One embodiment of the present invention provides a composition comprising an immune checkpoint inhibitor. A composition comprising an immune checkpoint inhibitor can attain a significant therapeutic effect at a high probability by administration thereof to a subject who has been selected by evaluation with the biomarker of the present invention.
The composition comprising an immune checkpoint inhibitor of the present invention is generally administered systemically or locally in an oral or parenteral form.
The dosage varies depending on the age, body weight, symptom, therapeutic effect, administration method, treatment time or the like, but is generally administered, for example, orally one to several times a day in the range of 0.1 mg to 100 mg per dose per adult, or is administered parenterally (preferably intravenously) one to several times a day in the range of 0.01 mg to 30 mg per dose per adult or continuously administered intravenously in the range of 1 hour to 24 hours per day. Of course, the dosage varies depending on various conditions, so that an amount less than the above dosage may be sufficient or an amount exceeding the range may be required.
For administration, a composition comprising an immune checkpoint inhibitor can have a dosage form such as a solid agent or liquid agent for oral administration or an injection, topical agent, or suppository for parenteral administration. Examples of solid agents for oral administration include tablets, pills, capsules, powder, granules and the like. Capsules include hard and soft capsules.
The composition of the present invention includes one or more active ingredients (e.g., antibody to an immune checkpoint protein), which is directly used or is mixed with an excipient (lactose, mannitol, glucose, microcrystalline cellulose, starch, or the like), binding agent (hydroxypropyl cellulose, polyvinyl pyrrolidone, magnesium aluminometasilicate, or the like), disintegrant (calcium cellulose glycolate or the like), lubricant (magnesium stearate or the like), stabilizer, solubilizing agent (glutamic acid, aspartic acid, or the like), which is formulated in accordance with a conventional method for use. The composition may also be coated with a coating agent (refined sugar, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose phthalate, or the like) or coated by two or more layers as needed. Capsules made of a substance that can be absorbed such as gelatin are also encompassed.
The composition of the present invention comprises a pharmaceutically acceptable aqueous agent, suspension, emulsion, syrup, elixir or the like when formulated as a liquid agent for oral administration. In such a liquid agent, one or more active ingredients is dissolved, suspended, or emulsified in a commonly used diluent (purified water, ethanol, a mixture thereof, or the like). Such a liquid agent may also contain a humectant, suspending agent, emulsifier, sweetener, flavor, fragrance, preservative, buffer, or the like.
Examples of injections for parenteral administration include a solution, suspension, emulsion, and solid injection that is used by dissolving or suspending it in a solvent at the time of use. An injection is used by dissolving, suspending or emulsifying one or more active ingredients into a solvent. Examples of solvents that are used include distilled water for injections, saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, combination thereof, and the like. Such an injection may also comprise a stabilizer, solubilizing agent (glutamic acid, aspartic acid, polysorbate 80™, or the like), suspending agent, emulsifier, analgesic, buffer, preservative, or the like. They are prepared by sterilizing or aseptic operation in the final step. It is also possible to manufacture an aseptic solid agent such as a lyophilized product, which is dissolved in sterilized or aseptic distilled water for injection or another solvent before use.
(Cancer)
Examples of target cancer in the present invention include, but are not limited to, melanoma (malignant melanoma), non-small cell lung cancer, renal cell cancer, malignant lymphoma (Hodgkin's or non-Hodgkin's lymphoma), head and neck cancer, urological cancer (bladder cancer, urothelial cancer, and prostate cancer), small cell lung cancer, thymic carcinoma, gastric cancer, esophageal cancer, esophagogastric junction cancer, liver cancer (hepatocellular carcinoma, intrahepatic cholangiocarcinoma), primary brain tumor (glioblastoma and primary central nervous system lymphoma), malignant pleural mesothelioma, gynecologic cancer (ovarian cancer, cervical cancer and uterine cancer), soft tissue sarcoma, cholangiocarcinoma, multiple myeloma, breast cancer, colon cancer and the like.
(Biomarker)
The present invention provides a novel biomarker for predicting a therapeutic effect of cancer immunotherapy. In one aspect, the T-cell composition of a subject is used as an indicator for predicting a therapeutic effect of cancer immunotherapy.
In one embodiment, a certain indicator of the T-cell composition of a subject at or greater than a response group threshold value indicates that the subject is a part of a response group to cancer immunotherapy. In another embodiment, a certain indicator of the T-cell composition of a subject at or less than a response group threshold value indicates that the subject is a part of a response group to cancer immunotherapy. In yet another embodiment, a certain indicator of the T-cell composition of a subject at or greater than an ineffective group threshold value indicates that the subject is a part of an ineffective group to cancer immunotherapy. In yet another embodiment, a certain indicator of the T-cell composition of a subject at or less than an ineffective group threshold value indicates that the subject is a part of an ineffective group to cancer immunotherapy.
Those skilled in the art can determine a suitable threshold value for each of such indicators. Those skilled in the art can predict a response to cancer immunotherapy of a subject at the indicated sensitivity and/or specificity by using the threshold value (ineffective group threshold value and/or response group threshold value) disclosed herein.
For the indicators disclosed herein, those skilled in the art can appropriately determine a threshold value that would achieve a desired sensitivity and specificity from results of determining an effect of cancer immunotherapy of a reference subject group. The group of subjects who are proven in the Examples of the present specification can be considered as a reference subject group. In other words, those skilled in the art can determine a threshold value from the results of experiments disclosed in the Examples or determine a new threshold value from results of a reference subject population upon practicing the present invention.
Sensitivity refers to the ratio of the number of subjects with a given feature in selected targets to the total number of subjects with the given feature in a population of subjects when selecting subjects with the given feature from among the population of subjects. For example, sensitivity is 100% when all subjects with a given feature in a population of subjects are selected. The sensitivity is 50% when half of subjects with a given feature in a population of subjects are selected. The sensitivity is 0% when none of the subjects with a given feature in a population of subjects are selected. Sensitivity is determined as, for example, (number of subjects with a given feature among selected targets)/(total number of subjects with a given feature in a subject population). Determination with high sensitivity means that when it is desirable to find a subject with a certain condition (e.g., ineffective group with respect to cancer immunotherapy), such a subject is likely to be definitively determined as being in such a condition.
The biomarker of the present invention which enables determination at high sensitivity is very useful for ensuring the discovery of an ineffective group with respect to a certain therapy. It is also possible to select a threshold value so that sensitivity would be high in accordance with such an objective.
Specificity refers to the ratio of the number of subjects with a given feature in selected targets to the total number of selected targets when selecting a subject with the given feature from among a population of subjects. For example, specificity is 100% when all candidates selected from among a subject population have the given feature. The specificity is 50% when half of the candidates selected from among a subject population have the given feature. The specificity is 0% when none of the candidates selected from among a subject population have the given feature. Specificity is determined as, for example, (number of subjects with a given feature among selected targets)/(total number of selected targets). Determination with high specificity means that the probability of incorrectly determining a subject who is not in a certain condition (e.g., response group with respect to cancer immunotherapy) as not being in such a condition (e.g., response group with respect to cancer immunotherapy) is low.
The biomarker of the present invention which enables determination with high specificity is useful, for example for preventing a determination that would incorrectly determine a response group to a certain therapy as an ineffective group to discontinue therapy. It is also possible to select a threshold value so that specificity would be high in accordance with such an objective.
For example, when identifying a subject as a part of an ineffective group with an indicator at or below a certain threshold value (ineffective group threshold value) when an increase of the indicator is correlated with the effect of cancer immunotherapy, subjects who are determined to be not a part of an ineffective group (i.e., stable group or response group) despite being a part of an ineffective group decreases (sensitivity increases) for threshold values that are set higher, but subjects who are determined as a part of an ineffective group despite not being an ineffective group (e.g., stable group or response group) increases (decrease in specificity). In contrast, subjects who are determined as a part of an ineffective group despite not being a part of an ineffective group (i.e., stable group or response group) decreases (specificity increases) for threshold values that are set lower, but subjects who are determined as not a part of an ineffective group (i.e., stable group or response group) despite being a part of an ineffective group increases (sensitivity decreases).
For the biomarker of the present invention, a threshold value can be set and used so that specificity and/or sensitivity is very high, so that the biomarker of the present invention can be used as an unprecedented and advantageous marker for predicting a therapeutic effect of cancer immunotherapy. Those skilled in the art can also suitably set a threshold value in accordance with the objective in such a range of threshold values at which both the specificity and sensitivity are very high. It should be understood that a proximate value of a specific value can be used as long as determination of interest can be performed even when a specific value is shown as an example of a threshold value.
The ratio of CD62Llow T-cells in CD4+ T-cells of a subject can be used as an indicator for predicting a response to cancer immunotherapy of the subject, e.g., as an indicator for selecting an ineffective group. The inventors have discovered that the ratio of CD62Llow T-cells in CD4+ T-cells higher than an ineffective group threshold value can predict at a very high precision that the subject is not a part of an ineffective group to cancer immunotherapy in such a case.
An ineffective group threshold value for the ratio of CD62Llow T-cells in CD4+ T-cells can be appropriately determined by those skilled in the art based on a reference, or a threshold value (Cutoff) shown in
For example, when using 19.4 as an ineffective group threshold value in the results of
When similarly using a value of 14.45 or less (e.g., 14.45, 13.8, 13.3, 12.3, or 10.9) as an ineffective group threshold value for the ratio of CD62Llow T-cells in CD4+ T-cells in the results of
When using a value of 22.55 or greater (e.g., 23.1, 24.1, 24.8, 25.05, 25.45, 25.95, 27, 28.75, or the like) as an ineffective group threshold value for the ratio of CD62Llow T-cells in CD4+ T-cells, an ineffective group can be predicted with the ratio as a biomarker with 100% sensitivity.
In other words, an ineffective group threshold value as a ratio of CD62Llow T-cells in CD4+ T-cells can be in the range of about 10 to about 30(%). Examples of such an ineffective group threshold value include about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30(%).
The ratio of Foxp3+CD25+ T-cells in CD4+ T-cells of a subject can also be used as an indicator for predicting a response to cancer immunotherapy of the subject, such as an ineffective group threshold value. The inventors have discovered that a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells in a subject derived sample lower than an ineffective group threshold value indicates that the subject is not a part of an ineffective group with respect to cancer immunotherapy.
The ineffective group threshold value for a ratio of Foxp3+CD25+ T-cells in CD4+ T-cells of a subject can be appropriately determined by those skilled in the art from a reference subject. Such an ineffective group threshold value can be in a range of about 2 to about 4(%). Examples of threshold values include about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0(%).
Indicators of the T-cell composition disclosed herein can be used in combination when preferred. Since the inventors have discovered that multiple indicators independently exhibit correlation with responsiveness, it is understood that multiple indicators, which are combined and used as an indicator for responsiveness, can further improve the precision of prediction.
When two or more indicators are combined as an indicator of responsiveness, an indicator represented by a formula using any number of variables can be used. When multiple indicators (X1, X2, X3 . . . Xn) are used, examples of indicators of responsiveness include but are not limited to the following:
F=a
1
X
1
b1
+a
2
X
2
b2
+a
3
X
3
b3
. . . +a
n
X
n
bn
F=X
1
c1
*X
2
c2
+X
3
c
3
. . . *X
n
cn
wherein each of a, b, and c is any real number. Responsiveness can be predicted from the magnitude of the indicator that is calculated by such a formula. Multivariate analysis by logistic regression or discriminant analysis can be performed on the novel indicators discovered by the inventors to determine a coefficient for use as an indicator of responsiveness to cancer immunotherapy of a subject.
While indicators that are combined are not limited, the inventors have discovered indicators such as the amount of a CD4+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response, amount of a dendritic cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response, the amount of a CD8+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response, amount of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells, and amount of an ICOS+CD62LlowCD4+ T-cell subpopulation. Indicators that predict responsiveness by different mechanisms can be combined for use as an indicator exhibiting stronger correlation with responsiveness, which is not false correlation.
For example, two or more indicator, such as three, four, five, or more indicators, that are selected from the group consisting of:
It is possible to show that a subject is not a part of an ineffective group with respect to cancer immunotherapy by combining indicators, as responsiveness to cancer immunotherapy. Similarly, it is possible to determine that a subject is a part of a response group with respect to response to cancer immunotherapy by combining indicators, as responsiveness to cancer immunotherapy.
Typically, responsiveness can be predicted by formula F(X, Y) using two indicators (X, Y) disclosed herein as variables. In some cases, a formula is a relative value of X to Y.
Any function of X and Y (F(X, Y)) can be used as a relative value of X to Y. Especially when it is understood that X is positively correlated with responsiveness and Y is negatively correlated with responsiveness, any function of X and Y (F(X, Y)), which monotonically increases with respect to X and monotonically decreases with respect to Y, can be used, but the formula is not limited thereto. With two or more variables representing responsiveness, a formula representing responsiveness can be found by regression from logistic regression or the like by calculating the contribution of each variable to responsiveness.
Examples of F(X, Y) representing responsiveness include, but are not limited to the following.
F=aX
r
+bY
s
F=X
r
*Y
s
wherein a, b, r, and s are any real numbers.
Integers can be used as r and s for simplicity of the formula. In some embodiments, examples of relative values of X to Y include, but are not limited to, Xn/Ym (n and m are any integer) such as X/Y and X2/Y.
When each of factors X and Y indicates responsiveness to therapy from different mechanisms, combination of such indicators can make the prediction of responsiveness more accurate. The study by the inventors shows that a formula with r and s in the range of −5 to 5 can be used to accurately predict responsiveness to cancer immunotherapy of a subject.
In one embodiment, a subject can be shown to be not a part of an ineffective group with respect to cancer immunotherapy by using the amount of T-cells correlated with a dendritic cell stimulation in an anti-tumor immune response as X and the amount of regulatory T-cells or a CD4+ T-cell subpopulation correlated with regulatory T-cells as Y. In this case, it is demonstrated that responsiveness to cancer immunotherapy of a subject can be accurately predicted using a formula with r and s in the range of −5 to 5. Examples of such a formula include X/Y, X2/Y, X3/Y, X4/Y, X5/Y, X/Y2, X2/Y2, X3/Y2, X4/Y2, X5/Y2, X/Y3, X2/Y3, X3/Y3, X4/Y3, X5/Y3, X/Y4, X2/Y4, X3/Y4, X4/Y4, X5/Y4, X/Y5, X2/Y5, X3/Y5, X4/Y1, X5/Y5, and the like.
Examples of the present specification show that F=X2.475/Y can be used as an indicator by logistic regression for a combination of the amount of CD62Llow T-cells in CD4+ T-cells of a subject (X) and the amount of Foxp3+CD25+ T-cells in CD4+ T-cells (Y), but those skilled in the art can appropriately derive a different combination or a different formula for the indicators disclosed herein by a similar analysis.
In regression analysis, a result from a sample greater than the number of combined variables +1 can be used to calculate a coefficient in a formula of a combination of variables. When a form of formula in a combination of two indicators is found by regression analysis, regression analysis is performed using a result in at least four samples. Preferably, regression analysis is performed using results in 20 or more samples. More preferably, regression analysis is performed using results in 30 or more samples. Regression analysis with a greater number of samples can be advantageous in that a combination of indicators that predicts responsiveness of a subject more accurately can be found.
In one embodiment, the amount of CD62Llow T-cells in CD4+ T-cells of a subject (X) and the amount of Foxp3+CD25+ T-cells in CD4+ T-cells (Y) can be used as an ineffective threshold value as a combined indicator. For example, a relative value of X to Y can be used as an indicator for predicting a response with respect to cancer immunotherapy of a subject.
For example, the present invention can calculate variables (X, Y), with a value selected from the group consisting of
The method of the present invention can also calculate variables (X, Y), with the amount of a regulatory T cell subpopulation or a CD4+ T cell subpopulation correlated with regulatory T cells as (Y). The method of the present invention can also calculate variables (X, Y), with a value selected from the group consisting of:
The present invention further provides a method of identifying a response group (PR) and stable group (SD) in a subject population determined not to be a part of an ineffective group using the above (X, Y). A method of identifying the response group (PR) and stable group (SD) can predict whether a subject is a part of the response group (PR) or stable group (SD) by calculate variables (Z, W), with
The relative value of X to Y is not particularly limited, but any function of X and Y (F(X, Y)), which monotonically increases with respect to X and monotonically decreases with respect to Y, can be used. For example, such a function can be
F(X,Y)=G(X)/H(Y); or
F(X,Y)=G(X)−H(Y)
wherein G(X) and H(Y) can be monotonically increasing functions with respect to X and Y, respectively. For example, G(X) can be XR, logR X, RX, or the like, wherein R is any real number satisfying the condition and is preferably a positive integer. For example, H(Y) can be YR, logR Y, or RY, or the like, wherein R is any real number satisfying the condition and is preferably a positive integer. In such a form, the accuracy of prediction can be improved by using a positive prediction for a therapeutic effect of cancer immunotherapy of X in combination with a negative prediction for cancer immunotherapy of Y as an indicator.
Examples of relative values of X to Y include, but are not limited to, Xn/Ym (n and m are any positive real numbers) such as X/Y and X2/Y. When each of factors X and Y indicates responsiveness to therapy from different mechanisms, combination of such indicators can make the prediction of responsiveness more accurate.
A function using Z and W is not particularly limited. Any function of Z and W (J(Z, W)) can be used. Examples of such a function can be
J(Z,W)=K(Z)*L(W); or
J(Z,W)=K(Z)+L(W)
wherein K(Z) and L(W) can typically be functions which monotonically increase with respect to Z and W, respectively. For example, K(Z) can be ZR, logR Z, RZ, or the like, wherein R is any real number satisfying the condition and is preferably a positive integer. For example, L(W) can be WR, logR W, RW, or the like, wherein R is any real number satisfying the condition and is preferably a positive integer. Based on J(Z, W), accuracy of determination of a response group (PR) and stable group (SD) in an ineffective group can be improved. Examples of relative values of Z to W include, but are not limited to, Zn*Wm (wherein n and m are any real number), such as W5*Z. When each of factors Z and W indicates responsiveness to therapy from different mechanisms, combination of such indicators can make the prediction of responsiveness more accurate.
When the amount of CD62Llow T-cells in CD4+ T-cells is X and the amount of Foxp3+CD25+ T-cells in CD4+ T-cells is Y, X/Y can be used as an indicator for predicting a response to cancer immunotherapy of a subject. The inventors have discovered that a subject with a high X/Y is shown to be not a part of an ineffective group with respect to cancer immunotherapy. Thus, a value of X/Y can be used as an ineffective group threshold value.
An ineffective group threshold value for X/Y can be appropriately determined by those skilled in the art based on a reference, or a value (Cutoff) shown in
When 7.35 is used as an ineffective group threshold value of X/Y, an ineffective group can be predicted using X/Y as a biomarker for determining whether a subject is a part of an ineffective group with sensitivity of 71.4% and specificity of 100%.
When a value of 7.35 or less (e.g., 7.35, 6.83, 6.31, 5.64, 5.01, or the like) is used as an ineffective group threshold value of X/Y, this can predict an ineffective group as a biomarker with specificity of 100%.
When a value of 9.305 or greater (e.g., 9.895, 10.19, 11.71, 12.07, 12.32, 12.42, or the like) is used as an ineffective group threshold value of X/Y, this can predict an ineffective group as a biomarker with sensitivity of 100%.
In other words, an ineffective group threshold value of X/Y can be in the range of about 5 to about 13. Examples of an ineffective group threshold value of X/Y include about 5, 6, 7, 8, 9, 10, 11, 12, and 13.
Furthermore, as a relative value of X to Y, X2/Y can be used as an ineffective group threshold value, which is an indicator for predicting a response to cancer immunotherapy of a subject. The inventors have discovered that a subject with high X2/Y is shown to be highly unlikely a part of an ineffective group with respect to cancer immunotherapy.
An ineffective group threshold value for X2/Y can be appropriately determined by those skilled in the art based on a reference, or a threshold value (Cutoff) shown in
When 174.3 is used as an ineffective group threshold value for X2/Y, X2/Y can predict an ineffective group as a biomarker for determining whether a subject is a part of an ineffective group with sensitivity and specificity both at 100%.
110.6, 118.2, 134.9, 151.6, 157.4, 174.3, 194.2, 202.3, 208.3, and the like can be used as other values of ineffective group threshold values for X2/Y.
In other words, an ineffective group threshold value for X2/Y can be in the range of about 110 to about 210. Examples of ineffective group threshold values for X2/Y include about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, and 210.
Those skilled in the art can use other relative values of X to Y by setting an appropriate threshold value at least from the data disclosed herein.
It is also possible to distinguish PR and SD (show that a subject is part of a response group) using results of calculating (e.g., multiplying) two or more indicators (biomarkers, BM) of the present invention. In one embodiment, a value of Zn*Wm (n and m are positive real numbers), with a first biomarker as “Z” and a second biomarker as “W”, can be used to distinguish PR and SD, but this is not limited thereto. It is also possible to use results from calculating (e.g., adding and/or multiplying) three or more biomarkers for distinguishing PR and SD. For example, when a PR group and an SD group are distinguished using the product W*Z of the ratio of CD25+Foxp3+CD4+ T-cells in CD4+ T-cells (W) and the ratio of ICOS+CD62LlowCD4+ T-cells in CD62LlowCD4+ T-cells (Z), it was demonstrated that a threshold value of W*Z of 1.816 can be used as a biomarker with sensitivity of 80% and specificity of 89.5% (bottom figure in the middle of
In addition, any function of Z and W (J(Z, W)) can be used as disclosed above. For example, a formula such as J=Zr*Ws with r and s in the range of −5 to 6 can be used to accurately predict the responsiveness to cancer immunotherapy of a subject. Examples of such a formula include Z*W, Z2*W, Z3*W, Z4*W, Z5*W, Z6*W, Z*W2, Z2*W2, Z3*W2, Z4*W2, Z5*W2, Z6*W2, Z*W3, Z2*W3, Z3*W3, Z4*W3, Z5*W3, Z6*W3, Z*W4, Z2*W4, Z3*W4, Z4*W4, Z5*W4, Z6*W4, Z*W5, Z2*W5, Z3*W5, Z4*W5, Z5*W5, Z6*W5, Z*W6, Z2*W6, Z3*W6, Z4*W6, Z5*W6, Z6*W6, and the like. Examples in the present specification show that Z*W5 can be used as a preferred prediction formula, which combines the ratio of CD25+Foxp3+CD4+ T-cells in CD4+ T-cells (W) and the ratio of ICOS+CD62LlowCD4+ T-cells in CD62LlowCD4+ T-cells (Z) by logistic regression or the like (
The present specification further provides an indicator that can be used to distinguish a response group (complete response+partial response) and stable group (intermediate group) among a subject population determined as not part of an ineffective group.
The ratio of Foxp3+CD25+ T-cells in CD4+ T-cells can be used as an indicator for predicting a response to cancer immunotherapy of a subject who has been predicted as not a part of an ineffective group. The inventors have discovered that a high ratio of Foxp3+CD25+ T-cells in CD4low T-cells in subjects shown as not a part of an ineffective group means that the subject is highly likely to be a part of a response group with respect to cancer immunotherapy. CD4+Foxp3+CD25+ T-cells are regulatory T-cells with an immunosuppressive property, so that it was unexpected to find that a subject with a high ratio of such cells is highly likely to respond to cancer immunotherapy.
A ratio of LAG-3CD62LlowCD4low T cell subpopulation in CD62LlowCD4low T-cells, or a ratio of PD-1+CD62LlowCD4low T cell subpopulation in CD62LlowCD4low T-cells can be used as an indicator for predicting a response to cancer immunotherapy of a subject who has been predicted as not a part of an ineffective group. The inventors have discovered that these cell subpopulations can be used to distinguish a response group (complete response+partial response) and stable group (intermediate group).
The method of the present invention can usebe used, for example, a comparison of a relative value of X to Y with a threshold value (ineffective group threshold value) comprising measuring the amount of CD8+ dendritic cells (X) and measuring the amount of a CD28+CD62LlowCD8+ T-cell (Y) as an indicator for predicting that the subject is not a part of an ineffective group with respect to the cancer immunotherapy.
It is possible to predict that a subject is not a part of an ineffective group with any biomarker disclosed herein combined with any ineffective group threshold value. In addition, it is possible to predict an ineffective group using a threshold value determined for such indicators as an ineffective group threshold value, and use a threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells for predicting that a subject population (preferably a subject population with an ineffective group excluded) is a part of a response group with respect to cancer immunotherapy as a response group threshold value.
Alternatively, a method of identifying a response group (PR) and a stable group (SD) in a subject population determined as not a part of an ineffective group is provided. A method of identifying a response group (PR) and a stable group (SD) can predict whether a subject is a part of a response group (PR) or a stable group (SD) by calculating variables (Z, W), with
A response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells can be appropriately determined by those skilled in the art based on a reference, or a result shown in
When 2.05 is used as a response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells, the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells can be used as a biomarker for predicting whether a subject is a part of a response group with sensitivity of 52.6% and specificity of 100%.
When a value of 2.05 or less (e.g., 2.05, 1.895, 1.76, 1.7, 1.61, or the like) is used as a response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells, this can be used for predicting a response group as a biomarker with specificity of 100%.
When a value of 3.35 or greater (e.g., 3.35, 3.63, 4.365, or the like) is used as a response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells, this can be used for predicting a response group as a biomarker with sensitivity of 100%.
In other words, a response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells can be in the range of about 1.6 to about 4.4(%). Examples of a response group threshold value for the ratio of Foxp3+CD25+ T-cells in CD4+ T-cells include about 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, and 4.4(%).
Another aspect of the present invention provides a method of applying cancer immunotherapy to a subject selected with any of the biomarkers disclosed above (preferably a combination of biomarkers). One embodiment provides a method of administering an immune checkpoint inhibitor to a subject in which any one of the above biomarkers is in a state indicated by any one of the threshold values disclosed herein.
(Fractionation/Separation of Cells)
A sample for fractionation/separation of T-cells can be suitably collected from a subject using a conventional method. For example, this can be collected from peripheral blood, bone marrow, tumor tissue, hematopoietic tissue, spleen, normal tissue, lymph, or the like of a subject. Sample collection from peripheral blood can be advantageous for being simple and non-invasive.
The composition of T-cells in a sample of a subject can be measured by those skilled in the art using a conventional method. Generally, the number of cells that are positive for a marker (e.g., CD4) defining a cell subpopulation of interest in a sample can be measured using flow cytometry or the like. Some embodiments of the present invention comprise measuring the amount of CD62Llow T-cells in CD4+ T-cells (X) and/or the amount of FoxP3+CD25+ T-cells in CD4+ T-cells (Y). The measurement of the composition of a cell population generally uses flow cytometry, but may use a method using an antibody array or immunostaining on a sample comprising cells, protein expression analysis in a sample comprising cells (e.g., western blot, mass spectrometry, HPLC, or the like), mRNA expression analysis in a sample comprising cells (microarray or next generation sequencing), or the like.
To measure the cell count in each cell subpopulation such as CD62LlowCD4+ T-cell subpopulation and CD4+CD25+CD4+Foxp3+CD25+ T-cell subpopulation, the measurement may be found by experimentally removing cells other than subpopulations of each kind from all cells. There is a kit for the materialization thereof. For example, cells corresponding to a CD4+CD62Llow T-cell subpopulation can be separated from peripheral blood without using a CD4 antibody or CD62L antibody when a CD4+ Effector Memory T cell isolation kit, human (Militenyi Biotech) is used. This is achieved by counting and recording the total live cell count, and counting and recording the number of cells obtained using this kit. When a CD4+CD25+ Regulatory T-cell isolation kit, human (Militenyi Biotech) is used, the cell count corresponding to a CD4+CD25+CD4+Foxp3+CD25+ T-cell subpopulation can be found without using an anti-FoxP3 antibody. Since FoxP3 is localized in the nucleus in cells, this has an advantage of eliminating a step for staining a molecule in the nucleus. As a similar kit, CD4+CD25+CD127dim/−/Regulatory T cell isolation kit, human (Militenyi Biotech) or CD25+CD49d− Regulatory T cell isolation kit, human (Militenyi Biotech) can also be selected.
An antibody does not need to be used. Antibodies that can specifically recognize and bind a molecule expressed on individual cells are prepared so that they can emit color when bound to a molecule expressed on the cell surface or inside the cells. The antibodies are then detected to measure the number of cells that are emitting color.
Since the molecules expressed on the cell surface or inside the cells are proteins, mRNA encoding a protein when the protein is expressed is also formed in the cells. In other words, it is sufficient to examine mRNA in individual cells to examine the presence/absence of mRNA encoding a protein of interest. This is made possible by a single cell gene expression analysis, i.e., mRNA analysis at a single cell level.
Examples of single cell gene expression analysis include 1) a method of next generation sequencing using Quartz-Seq, 2) a method of isolating cells using a Fluidigm C1 System or ICELL8 Single-Cell System to isolate cells and prepare a library with SMART-Seq v4, 3) a method of separating cells with a cell sorter and measuring the cells with quantitative PCR using an Ambion Single Cell-to-CT kit, 4) CyTOF SYSTEM (Helios), and the like.
In other words, blood is obtained, live cells are counted, and cells are separated with a cell sorter or the like. For example, Ambion Single Cell-to-CT kit can be used on the individual separated cells to measure the expression level of a specific gene with an apparatus for quantitative PCR. Based on the result, individual cells are examined as to which subpopulation among CD62LlowCD4+ T-cell subpopulation and CD4+Foxp3+CD25+ T-cell subpopulation the cells fall under, and the number of cells falling under each subpopulation is counted. The ratio of the numbers (ratio of x to y) is then found. Examples of candidate genes whose expression is examined 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, and the like.
As shown in the Example of the present specification, the genes whose expressions are increased in CD62LlowCD4+ T-cells compared to CD62LhighCD4 T-cells include: AURAKA, CCL17, CD101, CD24, FOXF1, GZMA, GZMH, IL18RAP, IL21, IL5RA, ND2, SMAD5, SMAD7, and VEGFA (
As shown in the Example of the present specification, the genes whose expressions are increased in CD62LhighCD4 T-cells compared to CD62LlowCD4+ T-cells include: BACH2, CCL28, CCR7, CD27, CD28, CD62L, CSNK1D, FOXP1, FOXP3, IGF1R, IL16, IL27RA, IL6R, LEF1, MAL, and TCF7 (
Measurement of the ratio of cell subpopulations or comparison with a threshold value in the present invention may use a reference sample with a defined signal. Signals between a reference prepared to induce a fluorescent signal corresponding to a given cell subpopulation (e.g., particle to which a fluorescent pigment is attached) and a sample comprising a cell population can be compared to measure the amount and ratio of a cell population in the sample by comparison with a reference. Signals between a reference prepared to induce a fluorescent signal corresponding to a predetermined threshold value (e.g., particle to which a fluorescent pigment is attached) and a sample comprising a cell population can also be compared to determine the presence/absence or the marker of the present invention in the T-cell composition in the sample by a comparison with a reference.
When a specific marker is determined to be high (high expression) or low (low expression) in the present invention, those skilled in the art can use a classification baseline for expression intensity that is commonly used in the art. For example, it is possible to divide CD62L into CD62Llow and CD62Lhigh using the signal intensity corresponding to a 10E2 signal when using a PE-labeled anti-human CD62L antibody as the boundary.
In one embodiment, CD62L can be determined as high (high expression) or low (low expression) as follows. An antibody which is used as a negative control of the same isotype of an anti-CD62L antibody is prepared. The antibody used as a negative control should not recognize (bind) any antigen on a T-cell, but may non-specifically adsorb thereto. For example, an antibody sold as an isotype control is used. The same fluorescent label is used for an anti-CD62L antibody and the negative control. After preparation, respective fluorescence patterns are overlaid. In a typical pattern, the isotype control has a peak at a portion with a low level of fluorescence while the anti-CD62L antibody has a peak where the fluorescence level is high, and the fluorescence level slowly decreases where the fluorescence is lower (
The determination of a boundary of low and high on the x axis (FL4-H) is now discussed. The right side of the figure is a schematic diagram when assuming that the peaks of low and high are divided. The peak of high appears to be horizontally symmetric, but low has a composite peak that cannot be considered horizontally symmetric. The peak of high is located at where FL4-H is, at about 400. The maximum amount of FL4-H of the peak of high (=A) is about 2,000. If the peak of high is considered horizontally symmetric, the inherent minimum amount of high (=B), which is separated by the same distance from FL4-H with the peak to A and is on the opposite side of the peak from A, is about 90. Up to this area, there should be an overlap with the peak of low. While the peak of high horizontally symmetric, horizontal symmetry is lost near D. In other words, D can be inferred to be the inherent maximum value of the peak of low, which means that there is a peak of low up to near D. Ultimately, high and low can be divided at the center of D, which is the maximum value of low, and B, which is the minimum value of high, i.e., C. This value corresponds to 10E2. In other words, the range of high can be C to A, and the range of low can be E to C. The area formed by the peak and each range corresponds to the cell count. The position of C on BD should vary depending on the ratio of sizes of peaks of high and low, sharpness of peaks or the like, but the difference from cases where the position of C is at the center of BD is considered small.
As used herein, “flow cytometry” refers to a technique of measuring the number of cells, individual or other biological particles suspended in a liquid and individual physical/chemical/biological attributes.
Various cells are analyzed using a flow cytometry technique. In particular, differentiation of blood cells can be determined using a flow cytometry technique. Such a determination of differentiation is starting to be used in diagnosis in addition to research.
Examples of advantages of flow cytometry include: the ratio accounted for blast cells can be readily found; specificity and sensitivity are high; it is highly reproducible; a large number of cells can be analyzed; the time required is short; and the like.
An apparatus using this technique is referred to as a “flow cytometer”. A flow cytometer is an equipment for measuring optical characteristics of a suspended matter (cell) from a homogeneous suspension of cells. Cells pass through the focal point of a laser beam on a liquid flow. When passing, optical characteristics of forward scatter, side scatter, and one or more fluorescent light with different wavelengths can be simultaneously measured for individual cells at 500 to 4000 cells per second to quickly and accurately measure biological characteristics such as the cell size, internal structure, the amount of nucleic acid or various antigens that are present in the cell membrane, cytoplasm, or the nucleus.
Scattered light is light scattered to the surrounding after a laser hits a cell. Forward scatter (FSC) is detected in front with respect to the laser optical axis. The scatter light intensity is proportional to the surface area of a cell. In other words, it is understood that a cell is large if the FSC value is relatively large, and the cell is small if the FSC value is small. Side scatter (SSC) is detected at a position that is 900 (perpendicular) to the laser optical axis. The scatter light intensity is proportional to the state of intracellular structure or cell granule. In other words, it is understood that the internal structure of a cell is complex if the SSC value is relatively large, and the internal structure of a cell is simple if the SSC value is small.
Results of flow cytometry can be typically expressed as a dot plot, with FSC as the X axis and SSC as the Y axis. Each cell is represented by a single dot (point) in the graph. The position thereof is determined by the relative values of FSC and SSC. Lymphocytes with a relatively small size and simple internal structure are displayed in the bottom left section, granulocytes with a large size and a granule inside are displayed in the top right section, and mononuclear cells with a large size but a simple internal structure are displayed between the lymphocytes and the granulocytes, as populations separated from one another.
Fluorescent light refers to a light generated when a fluorescent pigment labeling a cell is excited by an irradiated laser beam and releases energy. A flow cytometer (e.g., product name: Becton & Dickinson FACSCalibur) typically irradiates a 488 nm single wavelength laser beam and a 635 nm single wavelength laser beam. A cell itself also has a property of emitting weak fluorescence (autofluorescence). However, when actually attempting to specifically detect a molecule of a cell using fluorescence, it is necessary to bind a fluorescent pigment to a cell or its molecule in some form in advance. For example, FITC (Fluorescein isothiocyanate) absorbs a 488 nm excitation light and mainly emits a 530 nm fluorescent light (green). Labeling of an antibody with FITC in advance leads to a difference in the amount of antibodies bound in accordance with the amount of antigens on the cell surface, resulting in a difference in the fluorescence intensity of FITC. Thus, the amount of antigens that are present on the cell surface can be estimated. FACSCalibur that can be used as an example has four fluorescence detectors installed, which can detect different fluorescence wavelength regions. If multiple fluorescent pigments that emit light with different wavelengths are prepared, up to four different antigens can be simultaneously detected. As a fluorescent pigment other than FITC which is excited by a 488 nm single wavelength laser beam, PE (phycoerythrin) mainly emits a 585 nm fluorescent light, and PerCP (peridinin chlorophyll protein) and PE-Cy5 (carbocyanin-5) mainly emit a 670 nm fluorescent light. APC (allophycocyanin), which is a fluorescent pigment excited by a 635 nm single wavelength laser beam, mainly emits a 670 nm fluorescent light. These fluorescent pigments are combined with various antibodies and used in double or triple staining of cells. CD4, CD8, CD62L, CD25, Foxp3 molecules, and the like that are expressed on the surface of T lymphocytes can be detected with a monoclonal antibody, which specifically react thereto.
Strictly speaking, there are two types of flow cytometers, i.e., equipment which only analyzes cells and equipment capable of sorting analyzed cells. The latter is called “FACS”. As used herein, “FACS” is an abbreviation of fluorescence-activated cell sorter and refers to an apparatus used in a method of analyzing a surface antigen of a free cell such as a lymphocyte using a laser beam or sorting a specific cell by the presence/absence of a surface antigen or the like.
A result of flow cytometry can be displayed in a histogram, dot plot, or the like.
As used herein, “histogram” refers to a graph representing intensity of an optical signal of each parameter on the X axis and the cell count on the Y axis in fluorescence measurement using a flow cytometer. 10 thousand or more cells can be counted in total in such a form.
As used herein, “dot plot” refers to a plot of fluorescence intensity of two types of fluorescent pigments on the X and Y axes. In the double- or triple-stained analysis, this can be analyzed using a display method in which the respective fluorescence intensity is placed on the X or Y axis and individual cells correspond to each point on a two dimensional graph.
For example, peripheral blood or bone marrow liquid is collected, and then erythrocytes are removed by hemolytic method or specific gravity centrifugation, then the residual is reacted with a fluorescently labeled antibody (antibody to antigen of interest and a control antibody thereof) and sufficiently washed for observation using flow cytometry.
The detected scattered light or fluorescence is converted to an electric signal and analyzed by a computer. The result can distinguish lymphocytes, mononuclear cells, and granulocytes by representing the intensity of FSC as the cell size and the intensity of SSC as intracellular structure. The cell population of interest is gated thereafter as needed to examine the antigen expressing manner in the cells.
In practicing the method of the present invention, those skilled in the art can suitably identify a surface marker of the shown cells to fractionate or count the cells.
CD antigens were agreed upon in an international workshop to be classified (clustering) as clusters mainly by the biochemical feature (especially molecular weight) of an antigen recognized thereby. This is called CD classification. Many types of monoclonal antibodies that recognize a specific leukocyte differentiation antigen are named thereby under a unified convention, which is CD followed by a number, i.e., CD number (i.e., CD1, CD2, and the like).
Typical examples of cell surface markers including CD markers that are used herein are explained hereinafter.
CD4 (6.2): binds to an MHIC class II molecule on an antigen presenting cell and functions as a co-receptor of a T lymphocyte antigen receptor complex. CD4 is expressed in MHC class II restricted helper T lymphocyte.
CD8 (6.4): is a dimeric protein with an S—S bond of α and β chains. CD8 binds to an MHC class I molecule on an antigen presenting cell and functions as a co-receptor of a T lymphocyte antigen receptor complex. CD8 is expressed on MHC class I restricted killer T lymphocytes.
CD25: CD25 is a 55 kDa glycoprotein which is also known as a low affinity interleukin-2 receptor a chain (IL-2Rα). CD25 is also expressed in activated T-cells, B cells, and macrophages, and some non-activated CD4+ T-cells, which act as regulatory T-cells. Thus, CD25 is utilized as a marker for regulatory T-cells.
CD62L: CD62L (L-selectin) is a molecule, which is required for recognizing and homing a high endothelial venule (HEV) that is present specifically in lymphoid organs. Naive T-cells have this molecule to prepare for circulation through the lymphoid organs and antigen presentation. Naive T-cells lose a homing molecule upon recognizing an antigen presented by dendritic cells in lymphoid organs is recognized with a T-cell receptor and being primed by effector T-cells. Thus, effector T-cells that have been primed by antigen recognition and cloned and expanded have the CD62L° w phenotype.
Foxp3: Foxp3 is a master transcription factor of regulatory T-cells (Treg), i.e., transcription factor playing an essential role in all of differentiation/function expression/maintenance of differentiated status of Tregs. Since expression is nearly specific to Treg, Foxp3 is commonly used as a marker molecule for identifying Tregs. Foxp3 increases CD25 or CTLA4 expression, while suppressing the production of effector cytokines (IL-2, IFNγ, IL-4, IL-17, and the like).
PD-1 is deeply involved with the phenomenon of T cell exhaustion. In short, this phenomenon is attenuation of a T-cell reaction to antigens that are present in large quantity and for an extended period of time. Even if naive T-cells become T-cells with a high level of effector function due to priming by antigen presenting cells, the T-cells, upon exposure to large quantity antigen presentation for an extend period of time, express immune checkpoint molecules PD-1→LAG-3→CD244, lose the function and ultimately result in apoptosis. Since cancer cells are present “in large quantity” and “for an extended period of time”, it can be understood that this system is in effect.
(Effect of Preventing/Treating Cancer by CD62LlowCD4+ T-Cell Infusion)
Another aspect of the present invention is a method of improving or maintaining/sustaining a therapy effect of cancer immunotherapy by infusion of a specific cell or a composition therefor.
CD62LlowCD4+ T-cells have been found to be critical in a response of a subject to cancer immunotherapy. It is understood that the use of such T-cells can improve or maintain responsiveness to cancer immunotherapy of a subject. One embodiment of the present invention is a composition comprising a CD62LlowCD4+ T-cell. A CD62LlowCD4+ T-cell or a composition comprising the same is useful for concomitant use with cancer immunotherapy.
Although not wishing to be bound by any theory, a therapeutic effect due to CD62LlowCD4+ T-cell infusion into a patient on whom a PD-1 inhibitor and/or PD-L1 inhibitor does not achieve a sufficient effect of preventing/treating cancer can be understood as follows.
When PD-L1 expressed on the cancer cell surface binds to PD-1 expressed on the T-cell surface, an anti-tumor effect due to T-cells is suppressed (immune evasion mechanism by cancer cells). Anti-PD-1 antibodies are antibody molecules, which inhibit such a bond between PD-L1 and PD-1 and block the immune evasion mechanism by cancer cells to allow exertion of an anti-tumor effect by T-cells. Thus, it is understood that an anti-tumor effect due to inhibition of a PD-1/PD-L1 bond is primarily exerted in the effector phase where T-cells attack tumor, while the effect in the T-cell priming phase is low. In other words, it is difficult for a PD-1 or PD-L1 inhibitor to exert an anti-tumor effect unless there are already T-cell priming and sufficient effector T-cells. The maximum anti-tumor effect is exerted by anti-PD-1 antibodies on about 20% to 30% of cancer patients, but a T-cell immunity status required for an anti-PD-1 antibody to exert an anti-tumor effect and a method of evaluating such an immunity status were unknown.
CD62L (L-selectin) is a “homing receptor” of lymphocytes. CD62L is expressed on the cell surface of naive T-cells and promotes the migration thereof into the lymph node. When naive T-cells in the lymph node is subjected to antigen stimulation by an antigen presenting cell, the cells are activated into effector T-cells, while the CD62L expression level decreases (CD62Llow), and differentiate into CD4+ T-cells (helper T-cells) or CD8+ T-cells (cytotoxic T-cells). The inventors have discovered that CD62Llow T-cells are very effective as a method of identifying T-cells primed by cancer antigens under circumstances where cancer antigens are unknown and cancer antigen specific T-cells cannot be identified. In a mouse model, CD62Llow T-cells separated from a tumor regional lymph node can be adoptively infused to heal a cancer bearing mouse. Use of effector T-cells separated in this manner achieved a greater anti-tumor effect by introducing CD62LlowCD4+ T-cells (Example 4 and
The inventors have discovered that the ratio of CD62LlowCD4+ T-cells in all T-cells is clearly correlated with an anti-tumor effect of anti-PD-1 antibodies, i.e., it is essential to comprise CD62LlowCD4+ T-cells, which are T-cells exerting an anti-tumor effect, in order to exert an anti-tumor effect with anti-PD-1 antibodies.
Although not wishing to be bound by any theory, it is understood from this finding that T-cell immunity, which is ordinarily sufficient to exert an anti-tumor effect, is prepared, but immunity is evaded due to attenuation of antigen recognition signals by PD-1/PD-L1 in cancer patients comprising many CD62LlowCD4+ T-cells. CD62LlowCD4+ T-cells activate antigen presenting cells such as dendritic cells to activate a priming phase. Further, primed CD8+ T-cells need to be subjected to antigen presentation from local antigen presenting cells that have been activated by effector CD4+ T-cells in order to acquire a cytotoxic function. In this regard, it is understood that a PD-1/PD-L1 binding inhibitor, which restores attenuation of an antigen recognition signal primarily in the effector phase is complementary to the function of effector CD4+ T-cells. It is understood that the antigen presenting cell function, which should present cancer antigens, is still suppressed even if the immune evasion mechanism is blocked with an anti-PD-1 antibody in patients who do not comprise a large quantity of CD62LlowCD4+ T-cells, resulting in an unsatisfactory anti-tumor effect.
In view of the above, it is understood that administration of CD62LlowCD4+ T-cells can exert an anti-tumor effect with an anti-PD-1 antibody on patients with low CD62LlowCD4+ T-cell count resulting in an anti-tumor effect is not being exerted with an anti-PD-1 antibody.
(Manufacture and Use of Cell Containing Composition)
A method of manufacturing a composition comprising a CD62LlowCD4+ T-cell can comprising purifying CD62LlowCD4+ T-cells from a T-cell population derived from a human. The purifying may comprise removing a CD62L high expression cell from a T-cell population (negative selection) Purification of CD62LlowCD4+ T-cells by negative selection using an antibody and/or magnetic beads, or the like is preferable because impurities such as an antibody or magnetic beads do not remain on a cell to be used.
One embodiment of the present invention is a kit comprising a substance, which specifically binds to CD62L, for purifying CD62LlowCD4+ T-cells. Examples of substances which specifically bind to CD62L include, but are not limited to, antibodies that are specific to CD62L. Those skilled in the art can isolate and expand a specific T-cell subpopulation disclosed herein in accordance with a method disclosed herein, e.g., flow cytometry. In one embodiment, the composition disclosed herein provides a CD4+CD62Llow T-cell.
T lymphocytes can be collected in accordance with a known technique and concentrated or drained by a known technique such as flow cytometry and/or affinity binding to an antibody such as immunomagnetic selection. After the concentration and/or draining step, in vitro expansion of desired T lymphocytes can be performed in accordance with a known technique (including, but not limited to, the technique disclosed in U.S. Pat. No. 6,040,177 by Riddell et al.) or a variation thereof that will be apparent to those skilled in the art.
For example, a desired T-cell population or subpopulation may be expanded by adding a first T lymphocyte population to a medium in vitro, then adding a feeder cell to the medium (e.g., so that the produced cell population contains at least about 5, 10, 20, 40 or more feeder cells for every T lymphocyte in the first population to be expanded), and incubating the culture (e.g., for a sufficient time to increase the number of T-cells). The culture can be typically incubated under conditions, such as temperature, that are suitable for the expansion of T lymphocytes. For growth of human T lymphocytes, the temperature is generally, for example, at least about 25° C., preferably at least about 30° C., and more preferably about 37° C.
Cells can be separated and/or expanded, and then stored as needed and administered to a subject thereafter in accordance with the method disclosed herein or a method that is well known in the art.
The amount of cells of interested (e.g., CD62LlowCD4+ T-cell) in the composition comprising cell of the present invention, can be appropriately determined by the skilled in the art such that the intended effect is exerted, for example, may be at least 2×108, preferably at least 6×108, more preferably at least 2×109, for human administration.
A composition comprising cells disclosed herein can comprise a pharmaceutically acceptable carrier or excipient in addition to a cell of interest (e.g., CD62LlowCD4+ T-cell). As used herein, “pharmaceutically acceptable” means approved by a government supervisory authority or listed in the Pharmacopoeia or other commonly recognized pharmacopoeia for use in animals, or more specifically humans. As used herein, “carrier” refers to a culture, infusion solution, perfusate, diluent, adjuvant, excipient, or vehicle, which is administered with a therapeutic agent. The composition comprising cells of the present invention comprises cells as the main ingredient, so that a carrier can preferably maintain cells such as culture, infusion solution or perfusate. For example, when a pharmaceutical composition is intravenously administered, saline and aqueous dextrose are preferred carriers. Preferably, an aqueous saline solution and aqueous dextrose and glycerol solution are used as a liquid carrier of an injectable solution. When a medicament is orally administered, water is a preferred carrier. Examples of suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinylpyrrolidone, gelatin, middle chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, refined sugar, carboxymethylcellulose, cornstarch, inorganic salt, and the like. When desired, a composition can also contain a small amount of humectant or emulsifier, or a pH buffering agent. These compositions can be in a form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation or the like. A composition can also be formulated as a suppository by using a traditional binding agent and a carrier such as a triglyceride. An oral formulation can also comprise a standard carrier such as a medical grade mannitol, lactose, starch, magnesium stearate, saccharine sodium, cellulose or magnesium carbonate. Examples of suitable carriers are disclosed in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A). Such a composition contains a therapeutically effective amount of a therapeutic agent, preferably in a refined form, together with a suitable amount of carrier to be provided in a form that is suitable for administration to a patient. A formulation must be suitable to the manner of administration. A formulation may additionally comprise, for example, a surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, buffering agent, solubilizing agent, isotonizing agent, binding agent, disintegrant, lubricant, flow promoter, corrigent or the like.
One embodiment of the present invention is a method of using an amount selected from:
One embodiment of the present invention is a method of using a relative amount selected from:
One embodiment of the present invention is a method of using an amount selected from:
One embodiment of the present invention is a method of using a relative amount selected from:
One embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject using amounts (X, Y) selected from:
One embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject using amounts (X, Y) selected from:
Another embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject, wherein formula F(X, Y) higher than a threshold value (ineffective group threshold value) indicates that the subject is not a part of an ineffective group to the cancer immunotherapy. Formula F(X, Y) can be calculated, with a variable (indicator) of a formula for predicting a response to cancer immunotherapy of a subject, which is a value selected from the group consisting of:
Another embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject, wherein formula F(X, Y) higher than a threshold value (ineffective group threshold value) indicates that the subject is not a part of an ineffective group to the cancer immunotherapy. Formula F(X, Y) can be calculated, with a variable (indicator) of a formula for predicting a response to cancer immunotherapy of a subject, which is a value selected from the group consisting of:
Any function (F(X, Y)) of X and Y which monotonically increases with respect to X and monotonically decreases with respect to Y can be used as the aforementioned formula F(X, Y). Examples of formula F(X, Y) indicating responsiveness include F=Xr*Ys, wherein r and s are any real numbers. When X is positively correlated with responsiveness and Y is negatively correlated with responsiveness, it is preferable that r is a positive number and s is a negative number. Integers can be used for r and s for simplicity of the formula. For instance, F(X, Y) can be represented as Xn*Ym, wherein n and m are any integers. Examination of the inventors has shown that responsiveness to cancer immunotherapy of a subject can be accurately predicted using a formula with r and s in the range of −3 to 3. Examples of preferred forms of the formula include, but are not limited to, X/Y, X2/Y, X*Y, and the like.
One embodiment of the present invention is a method of using an amount selected from:
One embodiment of the present invention is a method of using a relative amount selected from:
One embodiment of the present invention is a method of using amounts (W, Z) selected from:
One embodiment of the present invention is a method of using amounts (Z, W) selected from:
Another embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject who has been determined to be not a part of an ineffective group, wherein formula J(W, Z) higher than a threshold value (response group threshold value) indicates that the subject is a part of a response group to the cancer immunotherapy. Formula J(W, Z) can be calculated, with variables (indicators) of a formula for predicting a response to cancer immunotherapy of a subject, which are:
Another embodiment of the present invention is a method of predicting a response to cancer immunotherapy of a subject who has been determined to be not a part of an ineffective group, wherein formula J(W, Z) higher than a threshold value (response group threshold value) indicates that the subject is a part of a response group to the cancer immunotherapy. Formula J(W, Z) can be calculated, with variables (indicators) of a formula for predicting a response to cancer immunotherapy of the subject, which are:
Any function (J(Z, W)) of Z and W which monotonically increases with respect to Z and monotonically increases with respect to W can be used as the aforementioned formula J(Z, W). Examples of formula J(Z, W) indicating responsiveness include J=Zr*Ws, wherein r and s are any real numbers. When Z is positively correlated with responsiveness and W is negatively correlated with responsiveness, it is preferable that r is a positive number and s is a negative number. Integers can be used for r and s for simplicity of the formula. For instance, J(Z, W) can be represented as Zn*Wm, wherein n and m are any integers. Examination of the inventors have shown that responsiveness to cancer immunotherapy of a subject can be accurately predicted using a formula with r and s in the range of −5 to 6. Examples of preferred forms of the formula include, but are not limited to, Z/W, Z2/W, Z*W, Z*W5 and the like.
The method disclosed herein, including those disclosed above, can be used to apply cancer immunotherapy to a subject who has been indicated as not a part of an ineffective group with respect to the cancer immunotherapy and/or as a part of a responsive group. Any cancer immunotherapy disclosed herein can be used.
One embodiment provides a composition for treating cancer in a subject who has been indicated as not a part of an ineffective group with respect to the cancer immunotherapy and/or as a part of a responsive group by using the method disclosed herein, including those disclosed above. The composition can comprise any active ingredient disclosed herein and have any constitution disclosed herein.
(General Techniques)
Molecular biological approaches, biochemical approaches, and microbiological approaches used herein are well known and conventional approaches in the art that are disclosed in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume], Idenshi Donyu Oyobi Hatsugen Kaiseki Jikken Ho [Experimental Methods for Transgenesis & Expression Analysis], Yodosha, 1997, and the like. The relevant portions (which can be the entire document) of the above documents are incorporated herein by reference.
As used herein, “or” is used when “at least one or more” of the listed matters in the sentence can be employed. When explicitly described herein as “within the range” of “two values”, the range also includes the two values themselves.
Reference literatures such as scientific literatures, patents, and patent applications cited herein are incorporated herein by reference to the same extent that the entirety of each document is specifically described.
As disclosed above, the present invention has been disclosed while showing preferred embodiments to facilitate understanding. The present invention is disclosed hereinafter based on Examples. The aforementioned description and the following Examples are not provided to limit the present invention, but for the sole purpose of exemplification.
Thus, the scope of the present invention is not limited to the embodiments and Examples specifically described herein and is limited only by the scope of claims.
The present invention is exemplified by the following Examples herein.
(1) [Example Demonstrating Enablement of the Invention of Markers]
Example 1: Therapeutic effect of Anti-PD-1 antibody and T-cell population composition
This Example demonstrates that analysis of CD62LlowCD4low T-cells and Foxp3+CD25+CD4+ T-cells using peripheral blood can predict a therapeutic effect of therapy with an anti-PD-1 antibody.
(2) [Example demonstrating enablement of invention of cell infusion]
Example 2: Hypothetical example of infusing CD62Llow cells
Example 3: Follow up observation
The Example demonstrates that an anti-tumor immune response of a patient is correlated with the T-cell composition, i.e., increase in CD62Llow cells enhances the anti-tumor immune response in a patient undergoing anti-PD-1 therapy to reduce tumor.
Example 4: Cell infusion into mice
The Example demonstrates that the percentage of CD62LlowCD4+ T-cell increases by infusing CD62LlowCD4+ T-cells in mice. An increase in the percentage of CD62LlowCD4+ T-cell and Treg also enhances anti-tumor immune responses in mice.
Example 5: Isolation/Expansion of CD62Llow cells
The Example demonstrates that CD62Llow cells can be successfully isolated and expanded.
1-1. Objective
The objective of this Example is to demonstrate that analysis of CD62LlowCD4+ T-cells and Foxp3+CD25+CD4+ T-cells using peripheral blood can predict a therapeutic effect of therapy with an anti-PD-1 antibody. The relationship between a therapeutic effect of an anti-PD-1 antibody and T-cell population composition was investigated.
1-2. Materials and Methods
The effect of nivolumab therapy in non-small cell lung cancer patients was studied in accordance with the protocol shown in
Peripheral blood was collected the day before nivolumab therapy from non-small cell lung cancer patients who have already undergone therapy.
CT was administered for determining the effect at week 8 from the start of nivolumab therapy. Partial response (PR), stable (SD), and progressive (PD) at this point were determined. The criteria of determination was in accordance with RECIST ver. 1.1. The following Table 1 shows the Characteristics of patients.
The composition of a peripheral blood T-cell population of subjects was analyzed as follows.
(1) Blood Collection
8 ml of blood was collected in a blood collecting tube for mononuclear cell separation (product name: BD Vacutainer® CPT™, BD Japan), which was gently inverted and mixed at room temperature.
(2) Centrifugation (Separation of mononuclear cells by specific gravity centrifugation) After blood collection, BD Vacutainer® CPT™ was centrifuged at 1500 to 1800×g for 15 minutes (centrifuge name/manufacturer: Kubota).
(3) Collection
About half of the plasma layer was aspirated so as not to disturb the cell layer above a gel barrier. The cell layer above the gel barrier was collected with a Pasteur pipette and transferred into a 50 ml tube (Falcon tube or the like). A phosphate-buffered balanced salt solution (10% FBS PBS) supplemented with 10% fetal bovine serum was added so that the mixture was 30 ml or greater. The mixture was centrifuged (4° C., 400 to 450 g×5 minutes) and washed twice.
(4) Cell Count
After completion of the first washing/centrifugation, 10 ml of PBS supplemented with 10% FBS (inactivated at 56° C. in 30 minutes) was added to resuspend cells. 50 μl of cell suspension in a centrifuge tube was collected. 0.1% trypan blue solution (50 μl) and the cell suspension were stirred. The cells were placed in an Improved Neubauer hemocytometer to count the cells.
(5) Freezing
After completion of the second washing/centrifugation, CELLBANKER™ 2 (Takara Bio) was used. Cells were resuspended in 5×105 to 5×106/ml, and transferred into a 2.0 ml cryogenic vial (Corning). After treatment, the cells were promptly frozen in a −80° C. deep freezer (Panasonic). After 24 hours and within one week of the above treatment, the cells were transferred into liquid nitrogen (under liquid phase).
(6) Culture
The frozen cells were adjusted to be 1 to 5×105/ml in an RPMI 1640 medium (FBS 10%) and cultured for 24 to 36 hours in a T-25 cell culture flask at 37° C. with 5% CO2.
(7) Cell Adjustment
Cell culture was collected in a 15 ml centrifuge tube and was centrifuged at 1500 rpm for 10 minutes to gather the cells at the bottom of the centrifuge tube. After the centrifugation, the supernatant was removed. 10 ml of FACS buffer was added to cell pellets to resuspend the cells with a pipette. The cells were against centrifuged at 1500 rpm for 10 minutes, and then the supernatant was aspirated. The cells were counted and adjusted so that the final cell concentration was 1.0×106 cells/ml. FACS buffer: 2% FBS, 0.05% Azide in PBS.
(8) Antibody Reaction
Suspension of peripheral blood mononuclear cells was placed in each FACS tube at 0.5 ml (5×105 cells would be in each tube). The tube was centrifuged with a centrifuge at 1500 rpm for 5 minutes. The cell pellets were left while aspirating and removing only the supernatant.
Tube 1
20 μl of FITC labeled anti-human CD4 antibody (25 μg/ml)
20 μl of PE labeled anti-human CD62L antibody (5 μg/ml)
20 μl of PE-Cy5 labeled anti-human CD8 antibody (5 μg/ml)
An antibody solution and cell suspension were stirred and mixed. The tubes were maintained at 4° C. After 30 minutes, 1 ml of FACS buffer was added to each tube with a Komagome pipette, and the mixture was centrifuged with a centrifuge at 1500 rpm for 5 minutes. The supernatant was aspirated and removed. 0.5 ml of 1% paraformaldehyde was added to each tube, from which the supernatant was aspirated while leaving only the cell pellets, to suspend the cells.
*Tube 2
20 μl of FITC labeled anti-human CD4 antibody (25 μg/ml)
20 μl of PE-Cy5 labeled anti-human CD25 antibody (5 μg/ml)
An antibody solution and cell suspension were stirred and mixed. The tubes were maintained at 4° C. After 30 minutes, 1 ml of FACS buffer was added to each tube, and the mixture was centrifuged with a centrifuge at 1500 rpm for 5 minutes. The supernatant was aspirated and removed. Intracellular Fixation and Permeabilization buffer Set™ (eBioscience) was used for cytoplasmic staining with 3 μl of PE labeled anti-human FOXP3 antibodies (500 μg/ml). 0.5 ml of 1% paraformaldehyde was added to each tube, from which the supernatant was aspirated while leaving only the cell pellets, to suspend the cells.
(9) Analysis by flow cytometry (product name: FACS Calibur™; BD Japan)
Measurement of Samples
Fluorescence of tubes 1 and 2 are measured.
Incorporation of analysis data for 30,000 cells
Analysis
STEP 1 Tube 1 is analyzed to identify a lymphocyte region using two-dimensional analysis using FSC or SSC. Cells gated in the lymphocyte region are further gated with respect to CD4+ fraction to obtain a histogram plot of CD62L (cell count in the blue region)
STEP 2 Tube 2 is analyzed to obtain two-dimensional analysis data with Foxp3 and CD25, which are gated in the lymphocyte region and CD4+ region.
(Cell Count in the Orange Region)
STEP 3 Calculation of the ratio of CD62LlowCD4+/Foxp3+CD25+CD4+ Formula Cell count in STEP 1/cell count in STEP 2
(Determination)
If lower than a predetermined value, Progressive Disease is predicted, for which drug is not effective.
If higher than a predetermined value, go to STEP 4
Step 4
Formula Cell count in the orange region/cell count for R1 and R2 in STEP 1×100(%)
(Determination)
If lower than a predetermined value, Stable Disease (SD) is predicted.
If higher than a predetermined value, Partial Response (PR) is predicted.
Statistical analysis was conducted on the relationship between the resulting T-cell population composition and observed therapeutic effect.
1-3. Results
The following Table 2 shows the observed therapeutic effect on patients.
Confirmed complete and partial responses were evaluated in accordance with the Response Evaluation Criteria in Solid Tumors, version 1.1 by the testers.
The ratio of therapeutic effect observed in this Example is approximately the same as the response rate obtained in the phase III clinical trial called checkmate 017. Thus, it is understood that there is no bias in a response to nivolumab.
As shown in
The result shows that the percentage of CD62low cells in CD8 cells was significantly lower in PD groups (
Furthermore, results of analyzing the correlation among three T-cell subpopulations with a difference between PD group and the PR+SD group are shown in panels D and E of
Furthermore, a correlation was not found between regulatory T-cells and the percentage of CD62LlowCD4+. This indicates that the respective cell counts are regulated by different mechanisms. It is understood that the precision of predicting a therapeutic effect can be enhanced by using both in combination as a biomarker.
A very good result of 92.6% sensitivity and 96.7% specificity was obtained with 19.4% as the threshold value, even by using only the percentage of CD62LlowCD4+ with a large difference (
The precision of prediction using a relative value of regulator T-cells and the percentage of CD62LlowCD4+ was examined.
For a formula using a combination of these factors, a suitable formula was examined using logistic regression from results in a sample of N=40 while considering the weighting for these factors with respect to the effect on therapeutic effects. A logistic regression model was used to find a coefficient, resulting in deriving the formula of X2.475/Y (
PR and SD were able to be identified with sensitivity of 52.8% and specificity of 100% with a threshold value of the percentage of CD25+Foxp3+CD4+ cells at 2.05% (
Although not wishing to be bound by any theory, the mechanism of predicting a clinical effect of cancer immunotherapy in the present invention can be understood as follows. *It is understood that CD4+ T-cells transmit an instruction to dendritic cells via an MHC class I molecule, and the dendritic cells receiving the instruction stimulate CD8+ T-cells via an MHC class II molecule. These CD4+ T-cells encompass effector T-cells (e.g., CD62LlowCD4+ T-cells) and regulatory T-cells (e.g., Foxp3+CD25+ T-cells). Meanwhile, the present invention predicts a clinical effect of cancer immunotherapy by evaluating the balance of both CD62LlowCD4+ T-cells and Foxp3+CD25+ T-cells. CD62L (L-selectin) is a molecule required for recognizing and homing a high endothelial venule (HEV) that is present specifically in lymphoid organs. Since naive T-cells, when stimulated by antigen presenting cells, are primed by effector T-cells so that CD62L expression decreases, homing is no longer performed by effector T-cells. Examples of markers of effector T-cell priming of naive T-cells include CCR7 as in CD62L. As a result of priming, the expression level of CCR7 decreases. Thus, CCR7 can be used instead of CD62Llow. For example, CCR7lowCD4+ T-cells and/or CCR7−CD4+ T-cells can be used instead of (or in addition to) CD62LlowCD4+ T-cells. Examples of cell subpopulations that can be used as an indicator of effector T-cells include, but are not limited to, subpopulations selected from the group consisting of CD62LlowCD4+ T-cell subpopulation, CCR7−CD4+ T-cell subpopulation, LAG-3+CD62LlowCD4+ T-cell subpopulation, ICOS+CD62LlowCD4+ T-cell subpopulation, CD45RA−CD4+ T-cell subpopulation, CD45RO+CD4+ T-cell subpopulation, HLA-DR+ dendritic cell subpopulation, CD80+ dendritic cell subpopulation, CD86+ dendritic cell subpopulation, PD-L1+ dendritic cell subpopulation, CD62LlowCD8+ T-cell subpopulation, and CD137+CD8+ T-cell subpopulation. The amount (absolute amount) and/or ratio (relative amount) of these cell subpopulations can be utilized as an indicator of effector T-cells. Examples of cell subpopulations that can be used as an indicator of regulatory T-cells include, but are not limited to, cell subpopulations selected from the group consisting of: an amount of a CCR4+CD25+CD4+ T-cell subpopulation; an amount of a CD62LhighCD25+CD4+ T-cell subpopulation; an amount of a CD127+CD25+CD4+ T-cell subpopulation; an amount of a CD45RA−Foxp3+CD4+ T-cell subpopulation; an amount of a CD4+Foxp3+CD25+ T-cell subpopulation; and an amount of an ICOS+CD62LlowCD4+ T-cell subpopulation. The amount (absolute amount) and/or ratio (relative amount) of these cell subpopulations can be utilized as an indicator of regulatory T-cells.
Before starting therapy with cancer immunotherapy, CD62LlowCD4+ T-cells are isolated from a peripheral blood sample of a subject and stored. The isolated CD62LlowCD4+ T-cells are expanded ex vivo (“ex vivo expansion” in
When a subject is determined as not a part of an ineffective group by the procedure shown in Example 1 or the like, cancer immunotherapy such as therapy using an anti-PD-1 antibody such as nivolumab is applied when the ratio of CD62LlowCD4+ T-cells/CD25+Foxp3+CD4+ T-cells is high, as shown in the top drawing of
In this regard, upon a decrease in an indicator such as CD62LlowCD4+ percentage/CD25+Foxp3+CD4+ cell percentage in the CD4+ T-cell composition of a subject to reach an immunological condition of an ineffective group, CD62LlowCD4+ T-cells expanded ex vivo can be infused to recover the original immunological condition to sustain the effect of cancer immunotherapy.
Storage/culturing cost can be minimized by culturing and infusing only CD62LlowCD4+ T-cells. This is more economical than continuing only an immune checkpoint inhibitor such as an anti-PD-1 antibody every two weeks.
A subject who has a low indicator such as CD62LlowCD4+ percentage/CD25+Foxp3+CD4+ cell percentage in the CD4+ T-cell composition of the subject and is determined as a part of an ineffective group (for example, low ratio of CD62LlowCD4+ T-cells/CD25+Foxp3+CD4+ T-cells as in the bottom drawing of
Seven patients were subjected to follow-up to observe the percentage of CD62LlowCD4+ T-cells. Peripheral blood mononuclear cells were analyzed every four weeks.
The results are shown in the following Table 3. Each of 1 to 7 represents a result for different patients.
Tumor regression was observed in patient 1 in the early stages of starting nivolumab therapy, but there was swelling in the cervical lymph node for a period of time. While PD was suspected, the swelling was reduced as of the evaluation CT after 8 weeks so that the patient was determined as PR. When an increase in tumor size was observed, the percentage of CD62LlowCD4+ T-cells decreased. When the tumor again regressed, the percentage of CD62LlowCD4+ T-cells was again elevated. All other subjects maintained a high percentage of CD62LlowCD4+ T-cells from before the therapy, so that they were determined as PR or SD.
Combined with the findings in Example 1, it is understood that a subject would be unresponsive when the percentage of CD62LlowCD4+ is less than 19.4%, but it is understood that a subject would be responsive when the percentage of CD62LlowCD4+ T-cells recovers again.
Tumor model mice were infused with cells having the composition of 2×106 CD62LlowCD4+/5×106 CD62LlowCD8+ (
The ratio of (CD62Llow cells in CD4+ T-cells)/(CD62LhighCD25+ cells in CD4+ T-cells) in the spleen on day 13 after tumor seeding was measured in a group infused with cells, i.e., 2×106 CD62LlowCD4+/5×106 CD62LlowCD8+ (
The above results show that an anti-tumor effect is achieved by infusing CD62LlowCD4+ cells and by infusing a mixture of CD62LlowCD4+ cells and CD62LlowCD8+ cells.
It can be understood that in a group infused with 1×106 CD62LlowCD4+ cells, the ratio of CD62Llow cells in CD4+ T-cells/CD62LhighCD25+ cells in CD4+ T-cells is high due to cell infusion at a stage where tumor regression has stopped (3.70→639.09), but the ratio of (CD62Llow cells in CD4+ T-cells)/(CD62LhighCD25+ cells in CD4+ T-cells) is reduced (4.55) when tumor again turns to an increase. This result shows that an effect of tumor regression is achieved by CD62Llow cells in a CD4+ T-cell population, not CD62L high expression cells such as CD62LhighCD25+ cells, and it is preferable to remove CD62L high expression cells from a cell containing composition achieving an anti-tumor effect.
CD62L staining patterns were observed for different races and mice.
Panel B (
6-1. Objective
The relationship between a therapeutic effect of an anti-PD-1 antibody and a marker expressed on a dendritic cell was investigated. It was examined whether a marker expressed on dendritic cells can be utilized in predicting a clinical effect of the cancer immunotherapy in the present invention.
6-2. Materials and Methods
Materials and methods are the same as Example 1. The antibodies shown in
6-3. Results
In view of the above results, the number/ratio of cells expressing HLA-DR and/or CD80 and/or CD86 in a myeloid dendritic cell (mDC) and/or plasmacytoid dendritic cell (pDC) population can be used as an indicator instead of (or in addition to) using CD4+ T-cells (CD62LlowCD4+ T-cells) as an indicator.
7-1. Objective
The relationship between a therapeutic effect of an anti-PD-1 antibody and a marker expressed on a CD8+ T-cell was investigated. It was examined whether a marker expressed on CD8+ T-cells can be utilized in predicting a clinical effect of tcancer immunotherapy in the present invention.
7-2. Materials and Methods
Materials and methods are the same as Example 1. The antibodies shown in
7-3. Results
Although not wishing to be bound by any theory, it is understood that (1) CD4+ T-cells transmits an instruction to dendritic cells via an MHC class I molecule, thus increasing dendritic cells expressing HLA-DR and/or CD80 and/or CD86 and (2) the dendritic cells receiving the instruction stimulate CD8+ T-cells via an MHC class II molecule, thus increasing CD62LlowCD137(4-1BB)*CD8+ T-cells, so that the number/ratio of CD62LlowCD137(4-1BB)*CD8+ T-cells and dendritic cells expressing HLA-DR and/or CD80 and/or CD86 can be used in predicting a clinical effect of cancer immunotherapy in the present invention in the same manner as the number/ratio of CD62LlowCD4+ T-cells.
Furthermore, exhaustion in this series of anti-tumor mechanisms recovers due to an anti-PD-1 antibody and an anti-PD-L1 antibody, while PD-1 expression on T-cells is not effective for the prediction of the effect of the present invention (data not shown). In view of this result, it is understood that PD-L1 expression on dendritic cells can also be used in predicting a clinical effect of cancer immunotherapy in the present invention.
8-1. Objective
It was examined whether markers other than CD62L expressed on CD4+ T-cells can be utilized in predicting a therapeutic effect.
8-2. Materials and Methods
Materials and methods are the same as Example 1. The antibodies shown in
8-3. Results
9-1. Objective
Example 1 demonstrated that the percentage of CD25+Foxp3+CD4+ cells is an excellent marker for separating PR and SD. Other markers for separating PR and SD were examined.
9-2. Materials and Methods
Materials and methods are the same as Example 1. The following markers using an antibody for detecting ICOS expressed on CD4+ T-cells were identical to the antibodies used in Example 8. The approach to determination is the same as Example 1.
9-3. Results
As is apparent from the results shown in
To derive a more detailed formula for an indicator, logistic regression was performed to further examine a formula combining the ratio of CD25+Foxp3+CD4+ T-cells in CD4+ T-cells (W) and the ratio of ICOS+CD62LlowCD4+ T-cells in CD62LlowCD4+ T-cells (Z).
As shown in
Because CD4+ T cells are critical to predict PD-1/PD-L1 blockade therapy NR, the inventors next examined whether PD-1 and LAG-3 expression on CD4+ T cells can be a marker distinguishing PR group and SD group, in addition to ICOS expression. The lymphocyte-activation gene 3 (LAG-3) protein, which is expressed on activated T cells, interacts with PD-1 to maintain T cell exhaustion. LAG-3 binds to MHC class II antigens and regulates the expanding effector T cell population size following antigen activation (28-30 Hui, E., et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science 355, 1428-1433 (2017); Baixeras, E., et al. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med 176, 327-337 (1992); Workman, C. J., et al. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J Immunol 172, 5450-5455 (2004)). The inventors thus examined PD-1, LAG-3, and ICOS expression on gated CD62Lhigh and CD62LlowCD4+ T cells.
The result is shown in
It is understood from the result of this Example, that the amount/ratio of LAG-3+CD62LlowCD4+ T-cell and PD-1+CD62LlowCD4+ T-cell subpopulation can be used as a marker to distinguish PR and SD, in addition to ICOS+CD62LlowCD4+ T-cell.
10-1. Summary
In order to demonstrate the prediction of therapeutic effect with the prediction formula (X2/Y, wherein X=the ratio of CD62Llow T-cells in the CD4+ T-cell population (%) and Y=the ratio of CD25+FOXP3+ T-cells in the CD4+ T-cell population (%)), the survival duration is analyzed in the discovery cohort (a portion of subject patient population of Example 1) whose treatment outcome (PD, SD, CR) have been determined. Further, in an independent validation cohort consisting of 41 patients continuing a treatment, whose prediction formulas were analyzed before assessment of tumor responsiveness, whether the prediction formula could distinguish NR (PD) was examined.
10-2. Materials and Methods
Characteristics of the patient group included in the discovery cohort and the validation cohort were as shown in the following table. The prediction formula values for each patient were calculated in accordance with the procedure described in Example 1.
10-3. Results
The prediction formula (X2/Y, wherein X=the ratio of CD62Llow T-cells in the CD4+ T-cell population (%) and Y=the ratio of CD25+FOXP3+ T-cells in the CD4+ T-cell population (%)) values for each patient in the discovery cohort are shown in
Next, the Inventors explored whether the prediction formula threshold value (X2/Y<192) could differentiate NRs in the independent validation cohort consisting of 41 consecutive patients whose peripheral blood was collected prior to Nivolumab therapy as the discovery cohort but was analyzed before tumor response evaluation. The prediction formula values were significantly higher (P=0.00068, t=3.693, df=39) in responding validation cohort patients as shown in
The objective responses at 8 week in each cohort were as follows.
The results in this Example showed that the method of predicting responsiveness to cancer immunotherapy described herein predicts accurately responsiveness to cancer immunotherapy, also in prospective study. Further, prediction of responsiveness to cancer immunotherapy also provides direct prediction of overall treatment response (overall survival (OS) or progression-free survival (PFS)) of patients.
Recently, it was demonstrated that CD28, but not the T cell receptor (TCR), is a primary target of PD-1-dependent signal inhibition. Thus, the Inventors examined if the percentage of CD28+ cells in the total population of CD8+ T cells correlated with the prediction formula value.
The Inventors found that the prediction formula (X2/Y, wherein X=the ratio of CD62Llow T-cells in the CD4+ T-cell population (%) and Y=the ratio of CD25+FOXP3+ T-cells in the CD4+ T-cell population (%)) values significantly (P=0.0045) correlated with the percentage of CD28+ cells in the total population of CD62LlowCD8+ T cell (
12-1. Summary
PBMC flow cytometry (FCM) analyses revealed that CD62LlowCD4+ T cell quantity and quality play a critical role in antitumor immunity and determine PD-1 blockade therapy response. The inventors performed microarray analysis to view CD62LlowCD4+ T cell differences at the molecular level among GR, IR, and NR patients in this Example. The Inventors first elucidated gene expression differences in CD62LhighCD4+ and CD62Llow CD4+ T cells. Then the differentially differential expressed gene on CD62Llow CD4+ T cells of the patients of each group was explored.
12-2. Materials and Methods
Total RNAs were isolated by TRIzol reagent (Thermo Fisher Science, Waltham, MA) from CD62Lhigh CD4+ and CD62LlowCD4+ T cells in PBMCs purified from two of each responder type. cDNA and cRNA synthesis and single-stranded cDNA (ssDNA) labeling reactions were performed according to the manufacture's instruction using the WT Plus Reagent Kit (Thermo Fisher Scientific). Total RNA (0.5 μg) was reverse transcribed into cDNA and subsequently synthesized into cRNA. ssDNA was reverse transcribed from 15 μg of cRNA and then labeled; 1.8 μg of labeled ssDNA was hybridized with microarray Clariom S assays for Human (Thermo Fisher Scientific) in a GeneChip Hybridization Oven 645. Hybridized arrays were scanned using the GCS3000 7G System (Thermo Fisher Scientific). The accession number ID of the gene expression data is GSE103157.
To identify the gene expression signature from two sets of gene expression data, the Inventors estimated the difference of gene expression between the two sets as follows. First, the Inventors performed the outlier test for all values of probes, and then calculated a z-score for each probe using the average and the variance of the probe values except for outliers. To compare two gene sets of z-scores, the z-score of each gene was transformed into probability, and then each difference of gene probability between the two sets, pd was calculated; i.e.,
where the k-th gene between the two gene sets, a and b, was compared. In this analysis, the Inventors selected the genes with
p
k
d>0.2 [Math. 2]
as the gene signature.
12-3. Results
For this, the Inventors first elucidated gene expression differences in CD62LhighCD4+ and CD62Llow CD4+ T cells. CD62LhighCD4+ T cells and CD62Llow CD4+ T cells have distinct gene expression profiles (
Then, the Inventors merged the genes in the signatures compared between the cells from GR and IR, GR and NR, IR and NR, GR+IR and NR, and GR and IR+NR (1884, 1826, 1410, 1167, and 1513 genes, respectively) (totaling 3484) (
As can be understood from the results of this Example, it is possible to determine a cell subpopulation that an obtained cell belongs to by examining the expression of differentially expressed genes between CD62LhighCD4+ and CD62LlowCD4+ T-cells, and it is thus possible to measure an amount and/or ratio of a cell subpopulation. Further it is understood that distinction of patients group can be achieved by examining the expression of differential expressed genes among respective patients groups on CD62LlowCD4+ T-cells.
Preparation of CD62Llow CD4+ T cells from tumor-draining lymph nodes 1.5×106 B16BL6 melanoma cells (in HBSS) was inoculated to B6 mice subcutaneously. Inguinal lymph nodes were harvested 9 to 10 days after. From harvested lymph nodes, CD4+ T-cells were isolated with CD4+ T-cell Isolation Kit+LS column. CD62LlowCD4+ T-cells were purified by negative selection with CD62L microbeads (LS column). These CD62LlowCD4+ T-cells were used for intravenous transfer.
Tumor Model
3×106 B16BL6 melanoma cells in HBSS were inoculated on midline of abdomen B6 mice subcutaneously. The mice were divide into (1) Control group (N=10), (2) Antibody group (N=17), and (3) Antibody+Cell group (N=4). No treatment was administered to Control group. After inoculating tumor cells to the Antibody+Cell group, the above CD62LlowCD4+ T-cells (1*106) were transferred on Day 4 or Day 5 after tumor inoculation, and anti-PD-1 antibody (RMP1-14 BioXcell 250 g) was administered intraperitoneally (on the day of cell administration, 3 days after and 6 days after cell administration). For the Antibody group, anti-PD-1 antibody (RMP1-14 BioXcell 250 g) was administered intraperitoneally (on the day of cell administration, 3 days after and 6 days after cell administration). The survival ratio of mice in each group was monitored.
Results
On day 16 after the administration of the antibody and/or T-cells, all individuals in Control group died, while the survival ratio of the Antibody+Cell group was 50%, which was higher than that of the Antibody group (
Anti-PD-1/PD-L1 antibodies are considered as primary therapy for almost all progressive cancer therapy. Meanwhile, the Ministry of Health, Labour and Welfare has warned that expensive drug costs can potentially increase the social security cost, so that the incremental cost-effectiveness ratio (ratio of increase in therapeutic effect to increase in drug cost) must be increased. The biomarkers provided in the present invention are medically and socially essential because they can predict an effect of an anti-PD-1/PD-L1 antibody in a simple, low cost, and accurate manner. The present invention is understood as a technique that is demanded worldwide for all cancers and tumors, thus having a very high market value.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-020685 | Feb 2017 | JP | national |
| 2017-110069 | Jun 2017 | JP | national |
The present application is a continuation of U.S. application Ser. No. 17/681,616, filed Feb. 25, 2022, which is a continuation of U.S. application Ser. No. 16/484,071 filed Aug. 6, 2019, now U.S. Pat. No. 11,293,924, which is a U.S. national phase application of PCT Application No. PCT/JP2018/004090 filed Feb. 6, 2018, which claims priority to Japanese Application Nos. 2017-110069 filed Jun. 2, 2017, and 2017-020685 filed Feb. 7, 2017. U.S. application Ser. No. 17/681,616 is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17681616 | Feb 2022 | US |
| Child | 18339853 | US | |
| Parent | 16484071 | Aug 2019 | US |
| Child | 17681616 | US |
