Methods and treatments

Abstract

The present invention relates to methods of treating cancer in a human subject that is non-responsive to immune checkpoint inhibitor treatment, methods of reducing the non-responsiveness of a subject to immune checkpoint inhibitor treatment, methods of determining whether a subject will be non-responsive to immune checkpoint inhibitor treatment, and compositions and kits for such uses.

Description

[0001] field

[0002] The present invention relates to methods of treating cancer in a human subject that is unresponsive to treatment with an immune checkpoint inhibitor, methods of reducing unresponsiveness of a subject to treatment with an immune checkpoint inhibitor, and compositions and kits for such uses.

[0003] background

[0004] Immune checkpoint therapy is a form of cancer treatment that involves modulating a patient's immune system to treat cancer by administering immune checkpoint inhibitors. Immune checkpoint inhibitors target checkpoint regulators, such as programmed cell death protein 1 (PD-1) or its ligand PD-L1, which inhibit the body's immune response against cancer cells.

[0005] Immune checkpoint inhibitors that have been approved and / or are in clinical trials for the treatment of cancer include the anti-PD-1 antibodies nivolumab (for metastatic melanoma) and pembrolizumab (for metastatic melanoma, lymphoma, mesothelioma, and non-small cell lung cancer), and the anti-PD-L1 antibodies avelumab (for urothelial carcinoma, Merkel cell carcinoma, renal cell carcinoma) and atezolizumab (for urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), small cell lung cancer (SCLC), and hepatocellular carcinoma (HCC).

[0006] However, a significant problem with treating cancer with immune checkpoint inhibitors is the large number of initial non-responders. For example, among patients with metastatic melanoma treated with anti-PD-1 antibodies, approximately 60%-70% do not respond to treatment.

[0007] WO 2019 / 051542 describes methods for identifying subjects who may respond to treatment with immune checkpoint inhibitors. While such methods identify and facilitate treatment of those who respond to checkpoint inhibitors, the treatment of cancer patients who do not respond to immune checkpoint inhibitors remains problematic.

[0008] What are needed are improved methods for treating cancer in patients who are unresponsive to immune checkpoint inhibitors.

[0009] Overview

[0010] The present inventors have discovered that a subpopulation of cells with high levels of intracellular arginase 1 plays an important role in the lack of responsiveness to immune checkpoint inhibitors targeting the interaction between PD-1 and its ligands.

[0011] As described in the Examples, the present inventors have discovered that non-responsiveness to immune checkpoint therapies targeting the interaction between PD-1 and its ligands is closely associated with an elevated population of a specific subpopulation of cells with high levels of intracellular arginase 1. Therefore, the present inventors contemplate that by inhibiting arginase 1 activity in a subpopulation of cells with high levels of intracellular arginase 1, a subject's non-responsiveness to immune checkpoint inhibitor therapy can be reversed, thereby allowing the subject to subsequently be effectively treated with an immune checkpoint inhibitor.

[0012] Thus, a first aspect provides a method of treating cancer in a subject that has been identified as unresponsive to immune checkpoint inhibitor therapy, comprising:

[0013] (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor; and

[0014] (b) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1;

[0015] The subject has been identified as unresponsive to the immune checkpoint inhibitor by:

[0016] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD 16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0017] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0018] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0019] A second aspect provides a method of treating cancer in a human subject, comprising:

[0020] (a) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, including:

[0021] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD 16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0022] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0023] (iii) comparing the distribution of the cells of the sample from the subject within the series of categories to the negative response control and / or the positive response control, wherein if the distribution of the cells of the sample from the subject within the series of categories is substantially the same as the distribution of cells represented by the negative response control within the series of categories and / or different from the distribution of cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment; and

[0024] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0025] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0026] A third aspect provides a method for increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to the treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a period of time sufficient for the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0027] (a) assessing the likelihood that the subject will not respond to immune checkpoint inhibitor treatment, including:

[0028] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the positive responders and the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers in each cell of the sample, such that cells expressing the same set of biomarkers are classified into the same category, wherein the set of biomarkers includes CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c , CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain and intracellular arginase 1;

[0029] (ii) determining the distribution of cells of a sample from the subject within the series of categories of the positive response control and the negative response control, the sample being in the form of a cell sample from the subject's blood;

[0030] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be likely to be non-responsive to the treatment.

[0031] In one embodiment, the panel of biomarkers includes at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase.

[0032] In one embodiment, the panel of biomarkers includes the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin beta 7.

[0033] In one embodiment, the panel of biomarkers includes at least 13 markers selected from the group consisting of CD45, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, GM-CSFR alpha chain, and intracellular arginase 1.

[0034] In one embodiment, the panel of biomarkers includes at least 13 markers selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), GM-CSFR alpha chain, and intracellular arginase 1.

[0035] In one embodiment, the panel of biomarkers includes CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

[0036] In one embodiment, the panel of biomarkers includes HLA-DR, CD14, CD16, CD66b, CD45RO, CD38, and intracellular arginase 1.

[0037] In one embodiment, the panel of biomarkers includes HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38, and intracellular arginase 1.

[0038] In one embodiment, the panel of biomarkers includes HLA-DR, CD14, CD16, CD66b, and intracellular arginase 1.

[0039] In one embodiment, step (ii) determining the distribution of cells of the sample within the set of categories comprises the following steps:

[0040] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0041] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0042] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0043] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0044] In one embodiment, step (iii) comparing the distribution of cells from a sample from the subject within the set of categories comprises the following steps:

[0045] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0046] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0047] In one embodiment, the series of categories includes: CD4+ naive Tcon, live CD4+ Treg, live CD4+ Tcon, CD45RO+CD4+ Tcon β7+, CD45RO+CD4+ Tcon CCR6+, live naive CD8+, live naive CD8+, CD8+(of)CD45RO+, live CD16 低 CD56 高 NK, live CD14+CD16- classical monocytes, live CD4+, live CD8+, naive CD8+.

[0048] In one embodiment, the series of categories includes the following categories: CD8+ Naive % Total, CD8+ % Total, CD8+CD45RO- Naive % Total, B Cells % Total, CD3+ % Total, CD4+ % Total, CD4 Naive % Total, CD4 Naive % CD4, CD4+ TCM % Total, Treg % Total, Treg CD45RO+ % Total, IntB7 % RO, CD8+CD45RO+ Memory % CD8, NK Cells % CD3-CD20-, CCR6 % RO, Monocytes % Total, CD14 Monocytes % Total, and NK Cells % Total. In one embodiment, the sample is a peripheral blood sample.

[0049] In one embodiment, the method further comprises monitoring the effectiveness of the treatment, comprising the step of comparing the level of cells expressing intracellular arginase 1 in a blood sample from the subject after treatment with the level of cells expressing intracellular arginase 1 before treatment.

[0050] In one embodiment, the panel of biomarkers includes a CD4 or CD8 T cell marker, and / or a B cell or plasma cell marker and / or a NK cell marker and / or a monocyte or dendritic cell marker, and at least 13 markers selected from the group consisting of: CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin beta 7.

[0051] A fourth aspect provides a method for treating cancer in a subject who is unresponsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor, and administering an effective amount of the immune checkpoint inhibitor, optionally in combination with additional administration of the agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0052] A fifth aspect provides a method of treating cancer in a human subject, comprising:

[0053] (a) assessing whether the subject will not respond to immune checkpoint inhibitor therapy, comprising determining whether a blood sample from the subject expresses intracellular arginase 1 (ARG1+) and is CD14 - 、CD16 - 、CD66b - The level of cells relative to the positive response control,

[0054] If the sample expresses intracellular arginase 1 (ARG1+) and is CD14 - 、CD16 - 、CD66b - If the level of cells expressing α1β1β2+ expressed in α1β1β2+ expressed in α1β2 ...

[0055] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0056] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0057] A sixth aspect provides a method for increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to the treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0058] determining the level of cells expressing intracellular arginase 1 (ARG1+) in a blood sample from the subject,

[0059] wherein the subject is determined to be non-responsive to the treatment if the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control.

[0060] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, and CD66b-.

[0061] In one embodiment, the cell expressing intracellular arginase 1 (ARG1+) is CD14-, CD16-, CD66b-, and HLA-DR-.

[0062] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16- and CD66b-, HLA-DR-, CD123+.

[0063] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, and CD38+.

[0064] In one embodiment, the cell expressing intracellular arginase 1 (ARG1+) is CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19-, and CD3-.

[0065] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+, and CD38+.

[0066] In one embodiment of the fifth or sixth aspect, the positive response control is the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- in a blood sample from a subject or more than one subject who responds to immune checkpoint inhibitor treatment.

[0067] In one embodiment, the immune checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand.

[0068] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0069] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

[0070] In one embodiment, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplizumab, durvalumab, atezolizumab, or avelumab.

[0071] In one embodiment, the agent targeted to a cell expressing arginase 1 targets a cell constitutively expressing arginase 1.

[0072] The seventh aspect provides a pharmaceutical composition comprising an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 and an immune checkpoint inhibitor.

[0073] An eighth aspect provides a kit for treating cancer, comprising:

[0074] (a) an agent that inhibits arginase 1 activity or targets cells expressing arginase 1; and optionally

[0075] (b) Immune checkpoint inhibitors.

[0076] A ninth aspect provides a method for assessing whether a subject suffering from cancer will not respond to treatment with an immune checkpoint inhibitor, the method comprising:

[0077] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of the one or more positive responders and the one or more non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by the cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes intracellular arginase 1 and at least four markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD1 4, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and GM-CSFRα chain;

[0078] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0079] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0080] In one embodiment, the panel of biomarkers includes intracellular arginase 1 and at least 8 markers selected from the group consisting of CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, and GM-CSFR alpha chain.

[0081] In one embodiment, the panel of biomarkers includes intracellular arginase 1 and at least 13 markers selected from the group consisting of: CD45, CD3, CD20, CD14, CD15, CD16, CD11b, CD19, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), and GM-CSFR alpha chain.

[0082] In one embodiment, the panel of biomarkers includes intracellular arginase 1 and CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, and CD56.

[0083] In one embodiment, the panel of biomarkers includes intracellular arginase 1 and CD3, HLA-DR, CD14, CD16, CD19, CD66b, CD45RO, and CD38.

[0084] In one embodiment, the panel of biomarkers includes intracellular arginase 1 and CD3, CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, and CD56.

[0085] In one embodiment, (ii) determining the distribution of cells of the sample within the set of categories comprises the following steps:

[0086] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0087] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0088] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0089] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0090] In one embodiment, (iii) comparing the distribution of cells from a sample from the subject within the set of categories comprises the following steps:

[0091] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0092] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0093] In one embodiment, the series of categories includes the following categories: CD8+ Naive % Total, CD8+ % Total, CD8+CD45RO- Naive % Total, B Cells % Total, CD3+ % Total, CD4+ % Total, CD4 Naive % Total, CD4 Naive % CD4, CD4+TCM % Total, Treg % Total, Treg CD45RO+ % Total, IntB7 % RO, CD8+CD45RO+ Memory % CD8, NK Cells % CD3-CD20-, CCR6 % RO, Monocytes % Total, CD14 Monocytes % Total, and NK Cells % Total.

[0094] A tenth aspect provides a method of determining whether a subject will not respond to treatment with an immune checkpoint inhibitor, comprising determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject;

[0095] Wherein if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responder control, then the subject is determined to be non-responsive to the treatment.

[0096] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-.

[0097] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+.

[0098] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, CD38+.

[0099] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19-, CD3-.

[0100] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+, CD38+.

[0101] In one embodiment, the immune checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand.

[0102] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0103] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

[0104] In one embodiment, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplizumab, durvalumab, atezolizumab, or avelumab.

[0105] In one embodiment, the blood sample is a peripheral blood sample.

[0106] In one embodiment, the cancer is non-small cell lung cancer (NSCLC) or colorectal cancer.

[0107] An optional first aspect provides an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject who has been identified as unresponsive to immune checkpoint inhibitor treatment;

[0108] or an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 in the preparation of a medicament for treating cancer in a subject who has been identified as unresponsive to treatment with an immune checkpoint inhibitor, wherein the subject has been identified as unresponsive to the immune checkpoint inhibitor by:

[0109] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, C D20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0110] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and / or the negative-responding control;

[0111] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0112] An optional second aspect provides an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject who has been identified as unresponsive to immune checkpoint inhibitor treatment;

[0113] or an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 in the preparation of a medicament for treating cancer in a subject who has been identified as unresponsive to treatment with an immune checkpoint inhibitor, wherein the subject has been identified as unresponsive to the immune checkpoint inhibitor by:

[0114] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a panel of biomarkers by cells of the sample such that cells with the same biomarker expression of the panel of biomarkers are classified into the same category, wherein the panel of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD1 9, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain and intracellular arginase 1;

[0115] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and / or the negative-responding control;

[0116] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0117] An optional third aspect provides an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for use in increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a cancer that is unresponsive to said treatment;

[0118] Use of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 in the preparation of a medicament for increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to said treatment, wherein the subject has been identified as being unresponsive to the immune checkpoint inhibitor by:

[0119] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, C D20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0120] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and / or the negative-responding control;

[0121] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0122] An optional fourth aspect provides an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject who is unresponsive to treatment with an immune checkpoint inhibitor; or use of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 in the preparation of a medicament for treating cancer in a subject in combination with an immune checkpoint inhibitor, wherein the subject is unresponsive to treatment with an immune checkpoint inhibitor.

[0123] An optional fifth aspect provides an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject that is unresponsive to treatment with an immune checkpoint inhibitor; or use of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 in the preparation of a medicament for treating cancer in a subject that is unresponsive to treatment with an immune checkpoint inhibitor in combination with an immune checkpoint inhibitor, wherein the subject has been identified as unresponsive to the immune checkpoint inhibitor by a method comprising the steps of:

[0124] (a) determining the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- relative to a positive responder control in a blood sample from the subject,

[0125] wherein the subject is determined to be non-responsive to the treatment if the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- in the sample is elevated relative to the positive responder control. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1: is a heat map showing the pre-treatment blood immune signature populations of 10 patients with NSCLC and a single healthy control. The data obtained by flow cytometry analysis and population identification using a panel of 13 mAbs (specific for CD45, CD3, CD4, CD8, CD45RO, CD197 (CCR7), CD25, CD127, CD196 (CCR6), integrin β7, CD56, CD16, and CD14) are analyzed in MeV. The columns show samples from each individual. The rows represent the measured parameters (% of cells in each subpopulation, and the rows are normalized to show the SD of the mean, as indicated by the top bar, with black indicating at least 1.5SD below the mean, and light gray indicating at least 2SD above the mean). 5 patients failed to produce a clinical response to anti-PD-L1 therapy (non-responders, indicated by black bars). 5 patients produced a clinical response to anti-PD-L1 therapy (responders, indicated by green bars). Hierarchical clustering using Pearson correlation and average linkage indicated that data from the five non-responder patients clustered together, as did data from the five responder patients, illustrating the utility of this panel of 13 mAbs.

[0128] Figure 2 A dot plot of mass cytometry analysis from a non-responder NSCLC patient is shown. Cells expressing arginase 1 (0.37% total) are gated (black polygons) and clearly distinguished from the remaining cells expressing lineage markers (CD3, CD14, CD16, CD66b, and CD19). Cells expressing arginase 1 are HLA-DR 低 、CD38 高 and CD45RO+.

[0129] Figure 3 is a graph showing the percentage of cells expressing intracellular arginase 1 in NSCLC as a function of clinical response to anti-PD-1 / PD-L1 therapy. **p<0.01.

[0130] Figure 4 Figure 1 shows the lack of correlation between plasma arginase or arginine and clinical response to anti-PD-1 / PD-L1 therapy in NSCLC. Left: Plasma arginase activity. Right: Plasma L-arginine. ns: Not significant.

[0131] Figure 5 It is a graph showing the correlation between plasma arginase activity and plasma arginine in NSCLC.

[0132] Detailed description

[0133] A major problem in treating cancer with immune checkpoint inhibitors is that a large number of subjects do not respond to treatment during the initial treatment period, and such subjects are referred to as initial non-responders. The present inventors have previously found that in non-responders, the main cell populations that are reduced are CD4 T cells (particularly regulatory T cells (Treg), naive conventional (i.e., non-regulatory) CD4 T cells (abbreviated herein as Tconv or Tcon), and central memory CD4 Tconv naive CD8 T cells) and NK cells, classical monocytes, and HLA-DR + Without wishing to be bound by theory, the inventors believe that the reduction in naive CD4 and CD8 T cells in non-responders is due to a systemic effect, as naive T cells circulate through the blood and secondary lymphoid tissues such as lymph nodes (LNs) and spleen, but are generally excluded from peripheral non-immune tissues such as tumors.

[0134] The number of immature T cells is regulated by thymic output and peripheral life span. Since the age of the cohort tested by the inventors is older, and thymic output is low in elderly subjects, the inventors believe that shortened peripheral life span (i.e., reduced survival) may be the possible direct cause of the reduction of immature CD4 and CD8 T cells. One of the most effective requirements for the survival of immature T cells is the low-level T cell receptor (TCR) signaling via the dendritic cells in the secondary lymphoid tissue. The peripheral T cells whose TCR expression is acutely eliminated die rapidly. The recognition of autoantigens in the secondary lymphoid organs produces a partial phosphorylation response that prolongs the survival of immature T cells.

[0135] The inventors believe that a reduction in TCR signaling would be expected to reduce both T cell survival and T cell responsiveness to antigens. The inventors believe that reduced T cell responsiveness to antigens would explain the failure of tumor-primed cells to mount an effective attack on tumors following administration of agents that interfere with the PD-1 / PD-L1 pathway, while reduced T cell survival would explain the accompanying defects in multiple T cell subsets.

[0136] A regulator of TCR signaling is the amino acid L-arginine. Many cancer patients have abnormally low plasma arginine concentrations. Regulators of systemic L-arginine levels include the enzymes arginase 1 and nitric oxide synthase, encoded by the genes ARG1 and NOS2.

[0137] Recent cancer immunity research has focused on the tumor microenvironment before treatment, where tumor-specific immune cells are significantly enriched compared to the circulation. In contrast, it has been reported that T cells responsible for tumor clearance in response to anti-PD-1 therapy have a different TCR repertoire than "exhausted" cells in the tumor microenvironment before checkpoint therapy. The present inventors believe that T cells responsible for tumor clearance in response to anti-PD-1 therapy must therefore respond to anti-PD-1 somewhere outside the tumor (most likely the tumor-draining lymph nodes) and then migrate into the tumor in response to therapy.

[0138] As described in the examples, the present inventors have analyzed peripheral blood mononuclear cells from non-small cell lung cancer patient cohorts before and after treatment with anti-PD-1 / PD-L1 immune checkpoint inhibitors. The present inventors have found that there is a statistically significant negative correlation between the number of cells expressing intracellular arginase 1 in the blood and the clinical response. The present inventors also found that the correlation between clinical response and plasma arginase and arginine is not statistically significant (although as expected, plasma arginase activity is higher in samples with lower arginine content). This indicates that cells with high intracellular arginase 1 are the main driving factors for T cell response downregulation, thereby damaging the response of T cells to immune checkpoint inhibitors to the extent that an effective clinical response cannot be established.

[0139] Therefore, the inventors propose that the use of agents targeting arginase 1 will increase responsiveness in subjects who are unresponsive to immune checkpoint inhibitors. Over time, this may restore the unresponsive immune profile to normal.

[0140] Thus, one aspect provides a method of treating cancer in a human subject, comprising:

[0141] (a) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, including:

[0142] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD 20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0143] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0144] (iii) comparing the distribution of the cells of the sample from the subject within the series of categories to the negative response control and / or the positive response control, wherein if the distribution of the cells of the sample from the subject within the series of categories is substantially the same as the distribution of cells represented by the negative response control within the series of categories and / or different from the distribution of cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment; and

[0145] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0146] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0147] In one aspect, a method of treating cancer in a human subject is provided, comprising:

[0148] (a) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, including:

[0149] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders or one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, C D20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0150] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and / or the negative-responding control;

[0151] (iii) comparing the distribution of the cells of the sample from the subject within the series of categories to the positive response control and / or the negative response control, wherein if the distribution of the cells of the sample from the subject within the series of categories is substantially the same as the distribution of cells represented by the negative response control within the series of categories and / or is different from the distribution of cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment; and

[0152] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0153] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0154] Another aspect provides a method of treating cancer in a subject that has been identified as unresponsive to treatment with an immune checkpoint inhibitor, comprising:

[0155] (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor; and

[0156] (b) thereafter administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1,

[0157] The subject has been identified as unresponsive to the immune checkpoint inhibitor by:

[0158] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD 20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0159] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0160] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0161] Another aspect provides a method of treating cancer in a subject that has been identified as unresponsive to treatment with an immune checkpoint inhibitor, comprising:

[0162] (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor; and

[0163] (b) thereafter administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1,

[0164] The subject has been identified as unresponsive to the immune checkpoint inhibitor by:

[0165] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, C D20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0166] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and / or the negative-responding control;

[0167] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

[0168] In one embodiment, step (ii) determining the distribution of cells of the sample within the set of categories comprises the following steps:

[0169] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0170] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0171] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0172] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0173] In one embodiment, step (iii) comparing the distribution of cells from a sample from the subject within the set of categories comprises the following steps:

[0174] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0175] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0176] Clinical response type

[0177] The first step of the method involves assessing whether a subject suffering from cancer known to be responsive to immune checkpoint inhibitor therapy is likely to be responsive or non-responsive to immune checkpoint inhibitor therapy.

[0178] A subject responded to immune checkpoint inhibitor treatment if they demonstrated a complete clinical response, partial clinical response, or stable disease.

[0179] A subject is considered unresponsive to immune checkpoint inhibitor therapy if the subject exhibits progressive disease.

[0180] As is commonly understood, a "complete response" to therapy is generally understood to mean the disappearance of all detectable signs of cancer that are responsive to treatment. A "partial response" is generally understood to mean a decrease in tumor burden in an individual, e.g., in terms of tumor number, size, and growth rate. A partial response may increase the time to disease progression. "Stable disease" is generally understood to mean neither sufficient tumor shrinkage to qualify as a partial response nor sufficient tumor growth to qualify as progressive disease.

[0181] In one embodiment, a complete clinical response, partial clinical response, stable or progressive disease may be determined by: RECIST 1.0 criteria (Therasse P, et al.) 2000 J. Natl Cancer Inst 92:2015-16 .

[0182] RECIST 1.0 criteria

[0183] A. Definition of Measurable and Non-Measurable Disease

[0184] Measurable disease: Presence of at least one measurable lesion.

[0185] Measurable lesions: Lesions that can be accurately measured in at least one dimension, where the longest diameter (LD) is:

[0186] ≥20 mm by conventional techniques (medical photographs [of skin or oral lesions], palpation, plain x-ray, CT, or MRI), or

[0187] ≥10 mm, using spiral CT scan.

[0188] Non-measurable lesions: All other lesions, including lesions too small to be considered measurable (<20 mm in longest diameter by conventional techniques or <10 mm in longest diameter by spiral CT scan), including bone lesions, leptomeningeal disease, ascites, pleural or pericardial effusions, cutaneous / pulmonary lymphangitis, unconfirmed abdominal masses not confirmed by subsequent imaging, cystic lesions, or disease documented only by indirect evidence (e.g., laboratory test values).

[0189] B. Measurement Method

[0190] Conventional CT and MRI: The minimum lesion size should be twice the reconstruction interval. The minimum baseline lesion size can be 20 mm, provided that images are reconstructed serially at a minimum of 10 mm. MRI is preferred, and when used, lesions must be measured in the same anatomical planes on subsequent examinations using the same imaging sequence. Whenever possible, the same scanner should be used.

[0191] Spiral CT: The minimum size of the baseline lesion can be 10 mm, provided that the images are reconstructed serially at 5 mm intervals. This standard applies to tumors of the chest, abdomen, and pelvis.

[0192] Chest X-ray: Lesions on chest X-ray are considered measurable when they are clearly defined and surrounded by aerated lung. However, MRI is preferred.

[0193] Clinical examination: According to RECIST criteria, clinically detected lesions are considered measurable only if they are superficial (e.g., skin nodules and palpable lymph nodes). In the case of skin lesions, color microphotography is required to document the size of the lesion, including a ruler in the field of view and the patient study number.

[0194] C. Baseline Recording of Target and Non-target Lesions

[0195] All measurable lesions (up to 5 lesions per organ and 10 lesions total, representing all involved organs) should be identified as target lesions and recorded and measured at baseline.

[0196] Target lesions should be selected based on their size (LD lesions) and their suitability for accurate repeated measurements (clinically or by imaging techniques).

[0197] The sum of LD for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as a reference to characterize objective tumor response.

[0198] All other lesions (or disease sites) should be identified as non-target lesions and should also be recorded at baseline. These lesions do not need to be measured, but the presence or absence of each lesion should be noted throughout follow-up.

[0199] Records of the index lesion or lesions should include the date of assessment, description of the lesion location, dimensions, and type of diagnostic study used to follow up the lesion or lesions.

[0200] All measurements should be made and recorded in metric notation using a ruler or calipers.

[0201] D. Response Standards

[0202] Disease assessments were performed every 6 weeks after initiation of treatment. However, subjects experiencing a partial or complete response must undergo a confirmatory disease assessment at least 28 days later. Assessments should be performed as close to 28 days later (if schedule permits), but no earlier than 28 days.

[0203] One or more target lesion response assessments were defined as follows:

[0204] Target lesion evaluation

[0205] Complete response (CR) - disappearance of all target lesions.

[0206] Partial response (PR) - a reduction of at least 30% in the sum of the LD of target lesions, with the baseline sum of the LD as reference.

[0207] Stable disease (SD) - neither sufficient shrinkage to qualify as a PR nor sufficient increase to qualify as progressive disease (PD), referenced by the smallest sum of LD since the start of treatment. Lesions, referenced by the smallest sum of LD recorded since the start of treatment or the appearance of one or more new lesions.

[0208] E. Evaluation of Non-target Lesions

[0209] The standard definitions used to determine objective tumor response in non-target lesions are as follows:

[0210] Complete response - disappearance of all non-target lesions.

[0211] Incomplete response / stable disease - persistence of one or more non-target lesions.

[0212] Progressive disease - the appearance of one or more new lesions and / or unequivocal progression of existing non-target lesions.

[0213] F. Evaluation of Overall Response Based on RECIST Response

[0214] Overall response is the best response recorded from the start of treatment until disease progression / recurrence is recorded. Generally, assignment of a subject to best response will depend on the achievement of measurement and confirmation criteria.

[0215] The table below presents the best overall response estimate for all possible combinations of tumor response in target and non-target lesions with or without new lesions.

[0216] target lesions Non-target lesions New lesions Overall response CR CR none CR CR Incomplete response / (SD) none PR PR No PD none PR SD No PD none SD PD any Yes or No PD any PD Yes or No PD any any have PD

[0217] NOTE: Subjects who experience a general deterioration in their health requiring discontinuation of treatment and who do not have objective evidence of disease progression at that time should be classified as “worsening.” Every effort should be made to document objective progression even after discontinuation of treatment.

[0218] In some cases, it may be difficult to distinguish residual disease from normal tissue. When the evaluation of a complete response relies on this determination, it is recommended that residual disease be examined (fine needle aspirate / biopsy) to confirm complete response status.

[0219] G. Confirmation Criteria

[0220] To be designated as PR or CR status, confirmatory disease assessment should be performed no less than 28 days after the response criteria are first met.

[0221] To be assigned SD status, follow-up measurements had to meet SD criteria at least once after study entry, with a minimum interval of 12 weeks.

[0222] Preparation of response controls

[0223] Methods disclosed herein may include another step: assessing one or more tissues or organs of an individual who has received an immune checkpoint inhibitor to determine the regression of the tumor in the individual, and therefore determining whether the individual is suitable for providing a positive or negative response control. In one embodiment, this step utilizes radiographic imaging to determine the position and volume of each of more than one tumor lesions in the subject after treatment with an immune checkpoint inhibitor. For example, this may involve recording a three-dimensional radiological image of the geographical location of each of more than one tumor lesions in the subject. Non-limiting examples of radiographic images that can be used to determine the position and / or volume of tumor lesions include positron emission tomography (PET) scans, x-ray computed tomography (CT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MRT) or a combination thereof.

[0224] Positive and / or negative response controls can be derived from a single individual. However, in some embodiments, it is preferred that the positive response control be derived from a cohort of positive responders or more than one positive responder. In some embodiments, the negative response control be derived from more than one non-responder.

[0225] As described herein, in the case of using a cohort of positive responders or more than one positive responder or non-responder to derive a response control, the control is based on the distribution data generated by the individual evaluation of each of the positive responder or non-responder samples selected for deriving the positive response control or negative response control. Thus, each sample is evaluated to determine the cell distribution profile specific to each sample. The cell distribution profiles of the responders are then compiled to form a positive response control, and the cell distribution profiles of the non-responders are compiled to form a negative response control.

[0226] This compilation can enable the identification of cell populations that are primarily associated with the lack of clinical response to therapy in the majority of non-responders.Relevant statistical methods are understood by those skilled in the art and are further described herein.

[0227] In one embodiment, the positive response control describes the cellular distribution of a positive responder sample before or after treatment with an immunomodulator. Preferably, the positive response control describes the cellular distribution of a positive responder sample before the positive responder undergoes treatment with an immunomodulator that results in a positive response.

[0228] In one embodiment, the negative response control describes the cell distribution of a sample of a non-responder before or after treatment with an immune checkpoint inhibitor. Preferably, the negative response control describes the cell distribution of a non-responder before the non-responder undergoes treatment with an immune checkpoint inhibitor that results in a negative response.

[0229] As described herein, the purpose of positive and negative response controls is to provide a reference point for which another individual is predicted for the possibility that a clinical response is not formed for treatment with the same immune checkpoint inhibitors (i.e., the same as for forming positive and negative response controls) for preferably the same cancer (i.e., the same as for treating in the individual from which positive and negative response controls are derived). Predicted based on the comparison between test sample (sample from subject) and positive and / or negative response controls. Comparison can be between the cell class or cell frequency in the subpopulation defined by positive and / or negative response controls and test sample.

[0230] In one embodiment, a response control can be identified according to the following steps:

[0231] (i) providing a responder cell sample in the form of cells from an individual who responds to immune checkpoint inhibitor therapy;

[0232] (ii) providing a non-responder cell sample in the form of cells from an individual who did not respond to immune checkpoint inhibitor therapy;

[0233] (iii) applying a cell distribution analysis to the responder cell sample, whereby cells in the responder cell sample are distributed into a series of classes based on the expression of a panel of biomarkers by each cell, such that cells expressing the same amount of each biomarker of the panel of biomarkers are classified into the same class, thereby forming a responder cell distribution profile;

[0234] (iv) applying the same cell distribution analysis to the non-responder cell sample, thereby forming a non-responder cell distribution profile;

[0235] (v) identifying cell distributions in the responder cell distribution profile that are not observed in the non-responder cell distribution profile, and / or cell distributions in the non-responder cell distribution profile that are not observed in the responder cell distribution profile,

[0236] The cell distribution in the responder cell distribution profile that is not observed in the non-responder cell distribution profile is identified as a positive response control, and / or the cell distribution in the non-responder cell distribution profile that is not observed in the responder cell distribution profile is identified as a negative response control.

[0237] Typically, a responder cell profile is a compilation of cell profiles from different responders. This increases the likelihood that the control contains a cell profile that correlates with response to therapy in a large proportion of individuals who respond or do not respond to therapy.

[0238] Typically, more than one non-responder cell sample can be a compilation of cell distribution profiles of different non-responders.

[0239] Cells can be distributed according to the expression of at least 5, 6, 7, 8, 9, 10, 11, 12, or at least 13 biomarkers, although in some embodiments the number of biomarkers can be 20, 30, 40, 100 or more.

[0240] The phrase "identical expression" and its grammatical variants in the context of "cells having identical expression" will generally be understood to refer to the level of identity or similarity between related subjects. The level of identity or similarity can be a function of the number of responder cell profiles that form a positive response control or the number of non-responder cell profiles that form a negative response control. In cases where the response controls are derived from a compilation of responder or non-responder cell profiles, it can be expected that "identical expression" refers to a level of identity between related subjects that is lower than what might be expected if the response controls were derived from a single responder or non-responder cell profile.

[0241] Biomarkers

[0242] In some embodiments, cells can be evaluated for the absence of expression of a biomarker, or the presence of expression of a biomarker. In some embodiments, cells can be evaluated for a specific expression level of a biomarker. For example, a cell can be described as having a "low" expression level of a biomarker (e.g., CD4 低 ) or “high” expression levels of biomarkers (e.g. CD4 高 ). The meaning of biomarker expression levels referred to as "high" or "low" is generally known to those skilled in the art. For example, CD4 高 and CD4 低 T cells or CD127 高 and CD127 低 The meaning of T cells is understood by the skilled artisan and is routinely determined using standard techniques.

[0243] For purposes of illustrating this approach only, in one example, cells can be analyzed to determine whether they can be distributed among the class CD25 + CD127 低 T reg In this example, the expression of proteins for CD25, CD127, and various other T regIn this example, an antibody specific for CD25 + CD127 低 T reg Cells with CD25 - CD127 低 T reg or CD25 + CD127 高 T reg or CD25 - CD127 高 T reg The cells were not distributed to CD25 + CD127 低 T reg Category.

[0244] Generally, biomarkers are molecules characteristically expressed by cells with immune functions (including lymphocytes, monocytes, granulocytes, etc.).

[0245] In one embodiment, the panel of biomarkers includes a CD4 or CD8 T cell marker and at least 13 markers, typically at least 14, 15, 16, 17, or 18 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, , FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0246] In one embodiment, the panel of biomarkers includes a CD4 or CD8 T cell marker and at least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27, or 28 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD2 7, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0247] In one embodiment, the panel of biomarkers includes a CD4 or CD8 T cell marker and at least 29 markers, typically at least 30, 31, 32, 33, 34, 35, 36, 37, or 38 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD2 7, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0248] In one embodiment, the panel of biomarkers includes CD4 or CD8 T cell markers and at least 39 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0249] In one embodiment, the panel of biomarkers includes molecules expressed by B cells or plasma cells.

[0250] In one embodiment, the panel of biomarkers includes a B cell or plasma cell marker and at least 13 markers, typically at least 14, 15, 16, 17, or 18 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, Ig D, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0251] In one embodiment, the panel of biomarkers includes a B cell or plasma cell marker and at least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27, or 28 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA -DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0252] In one embodiment, the panel of biomarkers includes a B cell or plasma cell marker and at least 29 markers, typically at least 30, 31, 32, 33, 34, 35, 36, 37, or 38 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA -DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0253] In one embodiment, the panel of biomarkers includes a B cell or plasma cell marker and at least 39 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, , FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0254] In one embodiment, the panel of biomarkers includes molecules expressed by NK cells.

[0255] In one embodiment, the panel of biomarkers includes an NK cell marker and at least 13 markers, typically at least 14, 15, 16, 17, or 18 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD , CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0256] In one embodiment, the panel of biomarkers includes an NK cell marker and at least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27, or 28 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA- DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0257] In one embodiment, the panel of biomarkers includes an NK cell marker and at least 29 markers, typically at least 30, 31, 32, 33, 34, 35, 36, 37, or 38 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA- DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0258] In one embodiment, the panel of biomarkers includes an NK cell marker and at least 39 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0259] In one embodiment, the panel of biomarkers includes molecules expressed by monocytes or dendritic cells.

[0260] In one embodiment, the panel of biomarkers includes a monocyte or dendritic cell marker and at least 13 markers, typically at least 14, 15, 16, 17, or 18 markers, selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, I gD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0261] In one embodiment, the panel of biomarkers includes a monocyte or dendritic cell marker and at least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27, or 28 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HL A-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0262] In one embodiment, the panel of biomarkers includes a monocyte or dendritic cell marker and at least 29 markers, typically at least 30, 31, 32, 33, 34, 35, 36, 37, or 38 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HL A-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0263] In one embodiment, the panel of biomarkers includes a monocyte or dendritic cell marker and at least 39 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD8 6, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0264] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin beta 7.

[0265] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of: CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin beta 7. In one embodiment, using this panel of biomarkers, the series of categories can be: CD45RO+CD8+; live CD14+, CD16-, classical monocytes; CD45RO+, CD4+Tcon CCR6+; live CD4+; live CD4+Tcon; live CD4+Treg; live CD8+; CD45RO+CD4+Tcon beta 7+; live naive CD8+; CD4+ naive Tcon; naive CD8+; and CD16 低 CD56 高 , living NK.

[0266] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of at least 13 markers selected from the group consisting of CD45, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, GM-CSFR alpha chain, and intracellular arginase 1.

[0267] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of intracellular arginase 1 and at least 4, 5, 6, 7, 8, 9, 10, 11, or at least 12 markers selected from: CD45, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, and GM-CSFR alpha chain.

[0268] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of at least 13 markers selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), GM-CSFR alpha chain, and intracellular arginase 1.

[0269] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of intracellular arginase 1 and at least 4, 5, 6, 7, 8, 9, 10, 11, or at least 12 markers selected from CD45, CD3, CD4, CD8, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), GM-CSFR alpha chain.

[0270] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

[0271] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

[0272] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD14, CD16, CD66b, CD45RO, CD38, and intracellular arginase 1.

[0273] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38, and intracellular arginase 1.

[0274] In one embodiment, the panel of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD14, CD16, CD66b, and intracellular arginase 1.

[0275] In one embodiment, a positive response control can represent the distribution of cells within a range of at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 types of cells.

[0276] In one embodiment, a negative response control can represent the distribution of cells across a range of at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 types of cells.

[0277] In one embodiment, the cells of the test sample are not assessed for T cell or B cell receptor diversity.

[0278] A positive response control can describe the distribution of cells of positive responders as the number of cells of each category as a percentage of the total number of cells. A negative response control can describe the distribution of cells of non-responders as the number of cells of each category as a percentage of the total number of cells. In one embodiment, where the checkpoint inhibitor is an anti-PD-1 antibody, the distribution of the cells assessed is at least 5, 6, 7, 8, 9 or at least 10 categories of cells according to Table 1 below.

[0279] In one embodiment, where the checkpoint inhibitor is an anti-PD-1 antibody, the cell distribution assessed is ARG1+, and at least 4, 5, 6, 7, 8, or at least 9 cell types according to Table 1 below.

[0280] Table 1

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] In the context of "determining whether the distribution of cells of the test sample within the series of categories of the negative response control is the same as the distribution of cells represented by the negative response control", the phrase "distribution is substantially the same" and its grammatical variations will generally be understood to refer to the level of similarity or identity between the test sample and the negative response control. Preferably, the distribution is the same in at least most categories of the negative response control. In some embodiments, there may be some differences in the distribution of a few categories of the negative response control.

[0287] In one embodiment, cells from responders and non-responders are obtained from peripheral blood. The cells can be enriched for leukocyte subsets. For example, the cells can be enriched for mononuclear or multinuclear cells.

[0288] According to the evaluation of cells for test samples described below and as described in WO2019 / 051542, the evaluation of cells that have been selected to form positive responders and / or non-responders for response controls can be performed by mass spectrometry flow cytometry methods (including TOF flow cytometry).

[0289] Determine the distribution of cells in a test sample (i.e., a sample from a subject)

[0290] The distribution of cells in a test sample can be determined according to the following steps:

[0291] a. evaluating the expression of each cell of the test sample for each biomarker in the panel of biomarkers for positive and / or negative response controls;

[0292] b. classifying each cell of the test sample into one of a series of categories of positive response control and / or negative response control;

[0293] c. Measuring the number of cells of the test sample in each category of the series of categories of the positive responding control and / or the negative responding control.

[0294] The evaluation of cell distribution in the test sample is the relative number of cells within each category of the series of categories taking into account the positive and / or negative response controls. This can also be expressed as the percentage of the number of cells of each category to the total number of cells in the test sample. These evaluations can be completed by mass spectrometry flow cytometry methods (including TOF flow cytometry), which enables individual cell evaluation for the expression of a large number of biomarkers (40 or more) for each cell. See: [ Spitzer MH and GP Nolan 2016 Cell 165:780-791 ].

[0295] Comparison of test samples and response controls

[0296] Determining whether the distribution of cells of the test sample within the series of categories of the negative response control is the same as the distribution of cells represented by the negative response control generally requires the following steps:

[0297] a. Comparing the measured number of cells of the test sample in each category of the series of categories of the negative response control to the number of cells of the negative response control in each category of the series of categories of the negative response control.

[0298] Determining whether the distribution of cells of the test sample within the series of categories of the positive response control is different from the distribution of cells represented by the positive response control generally requires the following steps:

[0299] b. Comparing the measured number of cells of the test sample in each category of the series of categories of the positive-responding control to the number of cells of the positive-responding control in each category of the series of categories of the positive-responding control.

[0300] These steps can be performed in silico using available software packages.

[0301] In one embodiment, the cell producing intracellular arginase 1 is a bone marrow cell. In one embodiment, the cell producing intracellular arginase 1 is CD14-, CD16-.

[0302] In one embodiment, the cell producing intracellular arginase 1 constitutively expresses arginase 1.

[0303] The term "determine" refers to "detecting" or "measuring" the level or presence or absence of an item to be detected (e.g., a cell type). As used herein, the term "level" refers to the amount, number, percentage or concentration of an item.

[0304] As mentioned above, the present inventors have observed that T cells that respond to immune checkpoint inhibitors are outside the tumor microenvironment. Therefore, the sample is a sample of tissue from outside the tumor microenvironment. In this regard, the sample is a blood sample. In one embodiment, the blood sample is a peripheral blood sample. The sample can be a blood sample enriched for mononuclear cells or multinuclear cells.

[0305] If a subject is assessed or identified as unresponsive to immune checkpoint inhibitor treatment, the unresponsiveness can be at least partially reversed by administering to the subject an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0306] Another aspect provides a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a cancer that is unresponsive to treatment with an immune checkpoint inhibitor, the method comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor.

[0307] Another aspect provides a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a cancer that is unresponsive to the treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a period of time sufficient for the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0308] (a) assessing the likelihood that the subject will not respond to immune checkpoint inhibitor treatment, including:

[0309] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the positive responders and the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by each cell of the sample, such that cells expressing the same set of biomarkers are classified into the same category, wherein the set of biomarkers includes CD4 or CD8 T cell markers, and / or B cell or plasma cell markers, and / or NK cell markers and / or monocyte or dendritic cell markers, and at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, H LA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0310] (ii) determining the distribution of cells of a sample from the subject within the series of categories of the positive response control and the negative response control, the sample being in the form of a cell sample from the subject's blood;

[0311] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be likely to be non-responsive to the treatment.

[0312] In one embodiment, the panel of biomarkers includes at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0313] Another aspect provides a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a cancer that is unresponsive to the treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a period of time sufficient for the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0314] (a) assessing the likelihood that the subject will not respond to immune checkpoint inhibitor treatment, including:

[0315] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and / or negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the positive responders and the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by each cell of the sample, such that cells expressing the same set of biomarkers are classified into the same category, wherein the set of biomarkers includes a CD4 or CD8 T cell marker, and / or a B cell or plasma cell marker, and / or a NK cell marker and / or a monocyte or dendritic cell marker, and at least 5 markers, typically at least 6, 7, 8, 9, 10, 11, 12 or at least 13 markers, selected from the group consisting of: CD45, CD45RA, CD45 RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1;

[0316] (ii) determining the distribution of cells of a sample from the subject within the series of categories of the positive response control and / or the negative response control, the sample being in the form of a cell sample from the subject's blood;

[0317] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be likely to be non-responsive to the treatment.

[0318] In one embodiment, the panel of biomarkers includes at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0319] In one embodiment, step (ii) determining the distribution of cells of the sample within the set of categories comprises the following steps:

[0320] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0321] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0322] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0323] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0324] In one embodiment, step (iii) comparing the distribution of cells from a sample from the subject within the set of categories comprises the following steps:

[0325] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0326] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0327] As mentioned above, the present inventors have found that cells in the blood of subjects that are associated with non-responsiveness to immune checkpoint therapy are ARG1 +The present inventors further characterized these cells and found that they are a unique cell population that exhibits a phenotype different from neutrophils, classical monocytes, non-classical monocytes, plasmacytoid dendritic cells, and basophils. In this regard, ARG1+ cells associated with non-responsiveness to immune checkpoint therapy include the phenotype ARG1+, CD14-, CD16-, CD66b-.

[0328] In one aspect, a method of determining whether a subject will not respond to treatment with an immune checkpoint inhibitor is provided, comprising determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject;

[0329] Wherein if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control, then the subject is determined to be non-responsive to the treatment.

[0330] Another aspect provides a method of treating cancer in a human subject, comprising:

[0331] (b) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, comprising determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject;

[0332] (c) wherein the subject is determined to be non-responsive to the treatment if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control; and

[0333] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0334] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0335] Additional aspects provide a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a cancer that is unresponsive to the treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a period of time sufficient for the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0336] determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject,

[0337] Wherein if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control, then the subject is determined to be non-responsive to the treatment.

[0338] In one embodiment, the positive response control is the level of ARG1+, CD14-, CD16-, CD66b- cells in a blood sample from a subject or more than one subject who responds to immune checkpoint inhibitor treatment.

[0339] In various embodiments, the ARG1+, CD14-, CD16-, CD66b cells have the following phenotype:

[0340] (a) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-;

[0341] (b) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+;

[0342] (c) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, CD38+;

[0343] (d) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+;

[0344] (e) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-;

[0345] (f) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-;

[0346] (g) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-;

[0347] (h) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33 低 ;

[0348] (i) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33 低 、CD192(CCR2) 中 ;

[0349] (j) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33 低 、CD45 低 ;

[0350] (k) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33 低 、CD45 低 ,CD56-;

[0351] (l) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33 低 、CD45 低 , CD56-, CD20-.

[0352] Additional aspects provide a method of treating cancer in a subject that is unresponsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1, for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor, for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor, and thereafter administering an effective amount of the immune checkpoint inhibitor, optionally in combination with additional administration of the agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0353] As used herein, the term "effective amount" refers to the dosage of a drug or other agent that is administered to produce a therapeutic effect. The exact dosage will depend on the purpose of treatment, and will be determined by those skilled in the art using known techniques (see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, ed., Lippincott, Williams & Wilkins). For example, an "effective amount" of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 will be an effective amount for making a subject respond to an immune checkpoint inhibitor.

[0354] An agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 is administered for a sufficient time for the subject to respond to an immune checkpoint inhibitor .

[0355] A subject's response to an immune checkpoint inhibitor can be assessed using, for example, the methods described in WP2019 / 051542.

[0356] In one embodiment, following treatment with an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1, a subject can be assessed for responsiveness to an immune checkpoint inhibitor by a method comprising:

[0357] determining the distribution of cells from a sample from a subject to whom the agent has been administered, in the form of a cell sample from the subject's blood, within the series of categories of a positive responding control and / or a negative responding control; and

[0358] The distribution of the cells from the sample from the subject within the series of categories is compared to the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the positive response control within the series of categories and / or different from the distribution of the cells represented by the negative response control within the series of categories, the subject is determined to be likely to respond to the treatment.

[0359] Therefore, the length of time required to administer an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 can be monitored by assessment at intervals following administration of the agent.

[0360] Agents that inhibit arginase 1 activity and / or target cells expressing arginase 1 are known in the art. Examples of such agents that inhibit arginase activity include, for example, N-hydroxy-L-arginine (NOHA), 2(S)-amino-6-borylhexanoic acid (ABH), N-hydroxy-nor-L-arginine (nor-NOHA), α-difluoromethylornithine (DFMO), L-norvaline, iodoacetyl-L-ornithine, iodoacetyl-L-lysine, L-lysine and L-citrulline, (2S)-(+)-amino-5-iodoacetylaminovaleric acid, NG-hydroxy -L-arginine, (2S)-(+)-amino-6-iodoacetamidohexanoic acid and (R)-2-amino-6-boryl-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, CB-1158 (numidargistat), OATD-02 (Patent No. WO2017191130A4), or compounds described in WO2019173188A1, WO2019177873A1 and WO2020161675A1.

[0361] In one embodiment, the agent inhibits intracellular arginase 1 and / or targets cells expressing high levels of intracellular arginase 1.

[0362] In one embodiment, the agent targeted to a cell expressing arginase 1 targets a cell constitutively expressing arginase 1.

[0363] In embodiments where the agent is targeted to cells expressing arginase, the agent can be an antibody that specifically binds to cells expressing arginase 1 and generally inactivates it.

[0364] In another embodiment, the agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 is an agent that reduces or inhibits arginase gene expression by inhibiting transcription or translation. For example, the agent that inhibits arginase activity can be, for example, a gene silencing nucleic acid that targets arginase mRNA, such as an siRNA agent (see, for example, WO00 / 44895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858), a CRISPR agent (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21; Qi et al., Cell. 2013 Feb. 28; 152(5):1173-83) or an antisense RNA.

[0365] Typically, the dosage range of the agent that inhibits arginase activity in ARG1+ cells or reduces or inhibits arginase gene expression that can be administered to a subject (preferably a human) is 1 μg to about 100 g per kilogram of the subject's body weight. The precise dosage administered will vary depending on many factors, including, for example, the type of cancer state being treated, the nature of the agent (e.g., small molecule, siRNA, or antibody), the metabolic stability and duration of action of the agent, age, weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, severity of the particular condition, and the subject's experience with therapy.

[0366] The dose can be administered at a frequency of several times per day, or it can be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. The frequency of the dose will be apparent to those skilled in the art and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0367] As used herein, "immune checkpoint inhibitors" are molecules or agents that inhibit immunosuppression through immune checkpoints. Checkpoint inhibitors can be inhibitors of any checkpoint that inhibits T cell function and is used for cancer treatment. In one embodiment, the checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand. In one embodiment, the checkpoint inhibitor that is an inhibitor of the interaction between PD-1 and its ligand is any anti-PD-1 or anti-PD-L1 antibody. Examples of anti-PD-1 antibodies used in cancer therapy include pembrolizumab, nivolumab, or cemiplizumab. Examples of anti-PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

[0368] In another embodiment, the checkpoint inhibitor is an inhibitor of the binding of CTLA-4 and its ligand. In one embodiment, the checkpoint inhibitor that is an inhibitor of the interaction between CTLA-4 and its ligand is an anti-CTLA-4 antibody. An example of an anti-CTLA-4 antibody used in cancer therapy is ipilimumab.

[0369] Other examples of checkpoint inhibitors include inhibitors of A2AR, CD276 (B7-H3), VTCN1 (B7-H4), IDO (indoleamine 2,3-dioxygenase), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin domain and mucin domain 3), and VISTA (V-domain Ig inhibitor of T cell activation).

[0370] Cancer can be any cancer that can be treated with immune checkpoint inhibitors in a subject to which such treatment is responded. Examples of cancers for which the methods described herein can be used for treatment include bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, urothelial carcinoma, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, squamous cell carcinoma, Merkel cell carcinoma, mesothelioma, non-small cell lung cancer (NSCLC), small cell lung cancer, colon cancer; Multiple myeloma (MM), Hodgkin's lymphoma (HL), B cell lymphoma or diffuse large B cell lymphoma (DLBCL).

[0371] Another aspect provides a pharmaceutical composition comprising an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier.

[0372] "Pharmaceutically acceptable carrier" means that it is compatible with the other ingredients of the composition and is not harmful to the subject. The composition may contain other therapeutic agents as described below and can be formulated, for example, by employing conventional liquid vehicles or diluents and pharmaceutical additives of a type suitable for the desired mode of administration (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) according to techniques such as those well known in the art of pharmaceutical formulation (see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005, Lippincott Williams & Wilkins).

[0373] Pharmaceutical compositions are generally in the form of sterile injectable aqueous suspensions. The suspension can be prepared according to known techniques and contains an active substance mixed with an excipient suitable for preparing an aqueous suspension. Such excipients may include suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersants or wetting agents may be naturally occurring phospholipids, for example, condensation products of lecithin or alkylene oxide with fatty acids such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols such as heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate.

[0374] Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, for example, as solutions in 1,3-butylene glycol. Water, Ringer's solution and isotonic sodium chloride solution are among the acceptable vehicles and solvents that can be adopted. In addition, sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, it has been found that fatty acids such as oleic acid can be used to prepare injectable preparations.

[0375] Another aspect provides a kit for treating cancer in a subject, comprising:

[0376] (a) an agent that inhibits arginase activity in ARG1+ cells or targets cells expressing arginase; and optionally

[0377] (b) Immune checkpoint inhibitors.

[0378] The kit may also include instructions for use.

[0379] In general, the term "treat" means affecting a subject, tissue, or cell to obtain a desired pharmacological and / or physiological effect, and includes: (a) preventing a disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating or ameliorating the effects of the disease, i.e., causing the effects of the disease to regress. In one embodiment, treatment results in a decrease in the number of cancer cells in the recipient subject.

[0380] The term "subject" refers to any animal suffering from a disease in need of treatment by the methods of the present invention. In the context of this disclosure, the subject is a human.

[0381] It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the invention, which will be limited only by the appended claims. As used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural indicators. Thus, for example, reference to an "antibody" includes more than one such antibody. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains.

[0382] Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are described herein.

[0383] All publications mentioned in this specification are incorporated herein by reference. It will be appreciated by those skilled in the art that many variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0384] In the appended claims and the foregoing description of the invention, unless the context requires otherwise due to explicit language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. specifying the presence of stated features but not excluding the presence or addition of further features in various embodiments of the invention.

[0385] In order to illustrate the nature of the present invention so that it may be more clearly understood, the following non-limiting examples are provided. Example

[0386] Peripheral blood samples are obtained using venipuncture and collected into tubes to prevent clotting (e.g., tubes containing EDTA or heparin). Leukocytes are purified using Ficoll-Hypaque density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs), which are then immediately prepared for flow cytometry or cryopreserved for long-term storage, e.g., at -80°C or in liquid nitrogen. Cryopreserved leukocytes are thawed prior to analysis using conventional techniques.

[0387] The distribution of cells is determined using techniques that identify the expression of a predetermined protein or transcript within each single cell within the sample. Typically, cells are treated with a mixture of between 10 and 40 antibodies, each specific for a separate cellular protein and labeled with a unique metal isotope or fluorescent dye, allowing the treated samples to be analyzed using mass cytometry or fluorescence flow cytometry, respectively.

[0388] In a cohort of 10 stage 4 non-small cell lung cancer (NSCLC) patients scheduled for anti-PD-1 / PD-L1 therapy, pre-treatment PBMCs were analyzed by mass cytometry. To determine the immune signature, data from a panel of 41 mAbs (antibodies specific for CD45, CD3, CD4, CD8, CD45RO, CD197 (CCR7), CD25, CD127, CD196 (CCR6), integrin β7, CD56, CD16, and CD14) were analyzed. Five patients responded clinically to anti-PD-1 / PD-L1 therapy, and in five patients, the disease progressed despite anti-PD-1 / PD-L1 therapy. The pre-treatment blood immune signature of anti-PD-1 / PD-L1 therapy is shown in Figure 1 Middle. Hierarchical clustering confirmed the utility of pretreatment blood features in distinguishing responders from non-responders.

[0389] Table 2 is Figure 1 Definition of cell populations depicted in Flow cytometric analysis using a panel of 13 mAbs (specific for CD45, CD3, CD4, CD8, CD45RO, CD197 (CCR7), CD25, CD127, CD196 (CCR6), integrin β7, CD56, CD16, and CD14) defined 14 cell populations that define the immune signature of clinical non-response to immunotherapy targeting the PD-1 / PD-L1 pathway.

[0390] Table 2:

[0391]

[0392] Intracellular detection of arginase 1 using a metal-conjugated mAb specific for human arginase 1 revealed that distinct populations of arginase 1 expressing cells were clearly identified within the total PBMC population, as shown in Figure 2. Figure 2 As shown in Figure 3, cells constitutively expressing high levels of arginase 1 in pre-treatment PBMCs from NSCLC patients were negative for HLA-DR, CD14, CD16, and CD66b, and positive for CD45RO and CD38. 低 CD14+) and neutrophils (PMN-MDSC, HLA-DR 低 Although M-MDSCs (CD16+CD66b+) have been reported to express arginase 1, no intracellular arginase 1 expression was detected in M-MDSCs or PMN-MDSCs in unstimulated, pre-treatment blood samples examined in this study. Therefore, the constitutive expression of intracellular arginase 1 in circulating blood cells cannot be explained by M-MDSCs or PMN-MDSCs described in previous studies.

[0393] In 24 NSCLC patients, a statistically significant inverse correlation was found between the number of cells expressing intracellular arginase 1 in PBMCs before treatment and the clinical response to anti-PD-1 / PD-L1 therapy ( Figure 3 ).

[0394] In a cohort of 21 NSCLC patients for whom pretreatment plasma samples were available, we measured plasma arginase activity (using a colorimetric assay that measures the conversion of L-arginine to L-ornithine) and plasma L-arginine (using mass spectrometry). There were no statistically significant differences between responder and nonresponder patients ( Figure 4), although, as expected, plasma arginase activity was higher in samples with lower arginine content ( Figure 5 These results strongly support a model in which intracellularly expressed arginase is a major driver of impaired T cell responses to anti-PD-1 / PD-L1 therapy, to the extent that effective clinical responses cannot be established.

[0395] These data indicate that therapies targeting arginase-producing cells rather than circulating arginase would be beneficial in converting non-responding patients to responder status.

[0396] Markers associated with cells expressing arginase 1

[0397] A panel of monoclonal antibodies was used to obtain a complete definition of the phenotype of cells expressing arginase 1 and how these cells differ from other circulating myeloid cells. The results are shown in Table 3.

[0398] Table 3: Differences in marker expression between arginase 1 expressing cells, neutrophils, classical monocytes and non-classical monocytes.

[0399]

[0400] Arginase 1 expression in myeloid-derived suppressor cells (MDSC) has been described in the literature and has been documented in both neutrophil PMN-MDSC and monocytic mo-MDSC. The cells expressing arginase 1 described herein are different from two types of MDSC: they are negative for CD14, which is used to identify mo-MDSC, and negative for CD15, CD16, and CD66b, which are used to identify PMN-MDSC. They express CD123, which is not expressed by MDSC. Plasmacytoid DC (pDC) also highly expresses CD123. The cells expressing arginase 1 described herein are different from pDC because the expression arginase 1 described herein is negative for HLA-DR expressed by pDC. Basophils also highly express CD123. The cells expressing arginase 1 described herein are different from basophils because basophils do not express arginase 1.

[0401] In a further experiment to evaluate the morphology of cells expressing arginase 1, PBMC expressing arginase 1 from NSCLC patients were sorted using a combination of mAbs for CD45, HLA-DR, CD11b, CD123, arginase 1, CD14, CD15, and lineage (CD19, CD3, CD56). For comparison, pDCs, neutrophils, and monocytes were also sorted. The sorted cells were centrifuged onto a slide and stained with Giemsa. Nuclear morphology analysis indicated that arginase 1-positive cells had an abnormal morphology, with multilobed nuclei, typically with 2 or 3 lobes. The nuclei of many cells expressing arginase 1 showed a brain-like appearance, while a few cells were cleaved in appearance. The cells did not contain the typical basophilic granules of basophils. A second population of CD123+HLA-DR-arginase 1-negative cells (0.23% of CD45+ cells) were smaller in size and had a lymphoid appearance. They may correspond to the ILC-like population described by Mora-Velandia et al., 2017, Front. Immunol. 8:176 in the context of psoriasis.

[0402] In addition to NSCLC, in which arginase 1-expressing cells are more common in the blood than in healthy controls and are associated with initial resistance to immunotherapy treatment targeting the PD-1 / PD-L1 pathway, the presence of abnormally high numbers of these cells has also been shown in colorectal cancer patients. Arginase 1-expressing cells were absent from cell mixtures of dissociated cancer samples from the same patients.

[0403] Targeting arginase 1-positive cells to restore and reverse non-responsiveness to anti-PD-1 / PD-L1 therapy

[0404] We propose that the use of agents targeting arginase 1-positive cells will allow the unresponsive immune signature to return to normal over time. However, if arginase 1-targeted therapy allows potential cancer-controlling T cells to resume normal signaling function even before other aspects of the immune signature (such as defects in naive T cells) have normalized, allowing sufficient time for the signature to recover before initiating anti-PD-1 / PD-L1 therapy may not be necessary.

[0405] This article also describes the following items 1 to 20:

[0406] 1. A method of treating cancer in a human subject, comprising:

[0407] (a) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, including:

[0408] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the one or more positive responders and the one or more non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 13 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD1 9, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1;

[0409] (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control;

[0410] (iii) comparing the distribution of the cells of the sample from the subject within the series of categories to the negative response control and / or the positive response control, wherein if the distribution of the cells of the sample from the subject within the series of categories is substantially the same as the distribution of cells represented by the negative response control within the series of categories and / or different from the distribution of cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment; and

[0411] (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and

[0412] (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0413] 2. The method according to claim 1, wherein the panel of biomarkers includes markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0414] 3. The method according to item 1 or 2, wherein (ii) determining the distribution of cells of the sample within the series of categories comprises the following steps:

[0415] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0416] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0417] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0418] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0419] 4. The method according to item 1, wherein (iii) comparing the distribution of cells in the sample from the subject within the series of categories comprises the following steps:

[0420] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0421] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0422] 5. The method according to any one of items 1 to 4, wherein the sample is a peripheral blood sample.

[0423] 6. The method according to any one of items 1 to 5, further comprising monitoring the effectiveness of the treatment, comprising determining a step of comparing the level of cells expressing intracellular arginase 1 in a blood sample from the subject after the treatment with the level of cells expressing intracellular arginase 1 before the treatment.

[0424] 7. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to said treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by:

[0425] (a) assessing the likelihood that the subject will not respond to immune checkpoint inhibitor treatment, including:

[0426] (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the positive responders and the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by each cell of the sample, such that cells expressing the same set of biomarkers are classified into the same category, wherein the set of biomarkers includes a CD4 or CD8 T cell marker, and / or a B cell or plasma cell marker, and / or a NK cell marker and / or a monocyte or dendritic cell marker, and at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, C D27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase;

[0427] (ii) determining the distribution of cells of a sample from the subject within the series of categories of the positive response control and the negative response control, the sample being in the form of a cell sample from the subject's blood;

[0428] (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be likely to be non-responsive to the treatment.

[0429] 8. The method according to item 7, wherein the panel of biomarkers comprises a CD4 or CD8 T cell marker, and / or a B cell or plasma cell marker and / or a NK cell marker and / or a monocyte or dendritic cell marker, and at least 13 markers selected from the following: CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0430] 9. The method according to item 7 or 8, wherein (ii) determining the distribution of cells of the sample within the series of categories comprises the following steps:

[0431] a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers for the response control;

[0432] b. classifying each cell of the sample from the subject into one of the series of categories of the response control;

[0433] c. measuring the number of cells of the test sample in each category of the series of categories in the response control;

[0434] The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

[0435] 10. The method according to item 7, wherein (iii) comparing the distribution of cells in the sample from the subject within the series of categories comprises the following steps:

[0436] comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control;

[0437] It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

[0438] 11. The method according to any one of items 7 to 10, wherein the sample is a peripheral blood sample.

[0439] 12. The method of any one of items 7 to 11, further comprising monitoring the effectiveness of the treatment, comprising determining the level of cells expressing intracellular arginase 1 in a blood sample from the subject after the treatment, compared to the level of cells expressing intracellular arginase before the treatment.

[0440] 13. A method of treating cancer in a subject that is unresponsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase activity or targets cells expressing arginase for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, and administering the effective amount of the immune checkpoint inhibitor, optionally in combination with additional administration of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

[0441] 14. The method according to any one of items 1 to 13, wherein the immune checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand.

[0442] 15. The method according to any one of items 1 to 14, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0443] 16. The method according to any one of items 1 to 15, wherein the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

[0444] 17. The method according to any one of items 1 to 16, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplizumab, durvalumab, atezolizumab or avelumab.

[0445] 18. The method according to any one of items 1 to 17, wherein the agent targeted to cells expressing arginase 1 targets cells constitutively expressing arginase 1.

[0446] 19. A pharmaceutical composition comprising an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 and an immune checkpoint inhibitor.

[0447] 20. A kit for treating cancer, comprising:

[0448] (a) an agent that inhibits arginase 1 activity or targets cells expressing arginase 1; and optionally

[0449] (b) Immune checkpoint inhibitors.

[0450] This application claims priority from Australian Provisional Patent Application No. 2022903578, the entire contents of which are incorporated herein by reference.

Claims

1. A method of treating cancer in a subject who has been identified as unresponsive to treatment with an immune checkpoint inhibitor, comprising: (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a sufficient period of time for the subject to respond to the immune checkpoint inhibitor; and (b) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1; The subject has been identified as unresponsive to the immune checkpoint inhibitor by: (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD 16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control; (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

2. A method of treating cancer in a human subject, comprising: (a) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, including: (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of one or more of the positive responders and one or more of the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least 5 markers selected from the group consisting of: CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD 16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain, and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control; (iii) comparing the distribution of the cells of the sample from the subject within the series of categories to the negative response control and / or the positive response control, wherein if the distribution of the cells of the sample from the subject within the series of categories is substantially the same as the distribution of cells represented by the negative response control within the series of categories and / or different from the distribution of cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment; and (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

3. The method of claim 1 or 2, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86 , FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase.

4. The method according to any one of claims 1 to 3, wherein the panel of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

5. The method of any one of claims 1 to 3, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, GM-CSFR alpha chain, and intracellular arginase 1.

6. The method of any one of claims 1 to 3, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), GM-CSFR alpha chain, and intracellular arginase 1.

7. The method of claim 1 or 2, wherein the panel of biomarkers comprises CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

8. The method of claim 1 or 2, wherein the panel of biomarkers comprises HLA-DR, CD14, CD16, CD66b, CD45RO, CD38, and intracellular arginase 1.

9. The method of claim 1 or 2, wherein the panel of biomarkers comprises HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38, and intracellular arginase 1.

10. The method of claim 1 or 2, wherein the panel of biomarkers comprises HLA-DR, CD14, CD16, CD66b, and intracellular arginase 1.

11. The method according to any one of claims 1 to 10, wherein (ii) determining the distribution of cells of the sample within the series of classes comprises the following steps: d. assessing the expression of each cell of the sample from the subject for each biomarker in the panel of biomarkers in response to the control; e. classifying each cell of the sample from the subject into one of the series of categories of the response control; f. measuring the number of cells of the test sample in each category of the series of categories in the response control; The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

12. The method according to any one of claims 1 to 11, wherein (iii) comparing the distribution of cells of the sample from the subject within the series of categories comprises the following steps: comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control; It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

13. The method according to any one of claims 1 to 6 or 11 or 12, wherein the series of categories includes CD4+ naive Tcon, live CD4+ Treg, live CD4+ Tcon, β7+ of CD45RO+CD4+ Tcon, CCR6+ of CD45RO+CD4+ Tcon, live naive CD8+, live naive CD8+, CD8+(of)CD45RO+, live CD16 低 CD56 高 NK, live CD14+CD16- classical monocytes, live CD4+, live CD8+, naive CD8+.

14. The method of any one of claims 1 to 6 or 11 to 12, wherein the serial categories include the following categories: CD8+ Naive % Total, CD8+ % Total, CD8+CD45RO- Naive % Total, B Cells % Total, CD3+ % Total, CD4+ % Total, CD4 Naive % Total, CD4 Naive % CD4, CD4+TCM % Total, Treg % Total, Treg CD45RO+ % Total, IntB7 % RO, CD8+CD45RO+ Memory % CD8, NK Cells % CD3-CD20-, CCR6 % RO, Monocytes % Total, CD14 Monocytes % Total, and NK Cells % Total.

15. The method according to any one of claims 1 to 14, wherein the sample is a peripheral blood sample.

16. The method of any one of claims 1 to 15, further comprising monitoring the effectiveness of the treatment, comprising the step of comparing the level of cells expressing intracellular arginase 1 in a blood sample from the subject after the treatment with the level of cells expressing intracellular arginase 1 before the treatment.

17. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to said treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by: (a) assessing the likelihood that the subject will not respond to immune checkpoint inhibitor treatment, including: (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from blood samples of the positive responders and the non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by each cell of the sample, such that cells expressing the same set of biomarkers are classified into the same category, wherein the set of biomarkers includes at least five markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, and CD11b , CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFRα chain and intracellular arginase 1; (ii) determining the distribution of cells of a sample from the subject within the series of categories of the positive response control and the negative response control, the sample being in the form of a cell sample from the subject's blood; (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be likely to be non-responsive to the treatment.

18. The method of claim 17, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86 , FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase.

19. The method of claim 17, wherein the panel of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin beta 7.

20. The method of claim 17, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, GM-CSFR alpha chain, and intracellular arginase 1.

21. The method of claim 17, wherein the panel of biomarkers comprises at least 13 markers selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), GM-CSFR alpha chain, and intracellular arginase 1.

22. The method of claim 17, wherein the panel of biomarkers comprises CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

23. The method of claim 17, wherein the panel of biomarkers comprises HLA-DR, CD14, CD16, CD66b, CD45RO, CD38, and intracellular arginase 1.

24. The method of claim 17, wherein the panel of biomarkers comprises HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38, and intracellular arginase 1.

25. The method of claim 17, wherein the panel of biomarkers comprises HLA-DR, CD14, CD16, CD66b, and intracellular arginase 1.

26. The method according to any one of claims 17 to 25, wherein (ii) determining the distribution of cells of the sample within the series of classes comprises the steps of: d. assessing the expression of each cell of the sample from the subject for each biomarker in the panel of biomarkers in response to the control; e. classifying each cell of the sample from the subject into one of the series of categories of the response control; f. measuring the number of cells of the test sample in each category of the series of categories in the response control; The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

27. The method of any one of claims 17 to 25, wherein (iii) comparing the distribution of cells from the sample of the subject within the series of categories comprises the steps of: comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control; It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

28. The method of any one of claims 17 to 27, wherein the series of categories comprises: CD4+ naive Tcon; live CD4+ Treg; live CD4+ Tcon; CD45RO+CD4+ Tconβ7+; CD45RO+CD4+ TconCCR6+; live naive CD8+; live naive CD8+; CD8+(of)CD45RO+; live CD16 低 CD56 高 NK; live CD14+CD16- classical monocytes; live CD4+; live CD8+; and naive CD8+.

29. The method of any one of claims 16 to 27, wherein the sample is a peripheral blood sample.

30. The method of any one of claims 17 to 29, further comprising monitoring the effectiveness of the treatment, comprising the step of determining the level of cells expressing intracellular arginase 1 in a blood sample from the subject after the treatment compared to the level of cells expressing intracellular arginase before the treatment.

31. A method of treating cancer in a subject that is unresponsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase activity or targets cells expressing arginase, for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, and administering the effective amount of the immune checkpoint inhibitor, optionally in combination with additional administration of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

32. A method of treating cancer in a human subject, comprising: (d) assessing whether the subject will not respond to immune checkpoint inhibitor treatment, comprising determining the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- in a blood sample from the subject; (e) wherein the subject is determined to be non-responsive to the treatment if the level of cells expressing intracellular arginase 1 (ARG1+) in the sample is elevated relative to a positive response control; and (b) if the subject is determined to be unresponsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1 for a period of time sufficient to allow the subject to respond to the immune checkpoint inhibitor; and (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with additional administration of said agent that inhibits arginase 1 activity and / or targets cells expressing arginase 1.

33. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from cancer that is unresponsive to said treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 for a sufficient period of time to allow the subject to respond to the immune checkpoint inhibitor, wherein the subject has been determined to be unresponsive to the immune checkpoint inhibitor by: determining the level of cells expressing intracellular arginase 1 (ARG1+) in a blood sample from the subject, wherein the subject is determined to be non-responsive to the treatment if the level of cells expressing intracellular arginase 1 (ARG1+) and being CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control.

34. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, and CD66b-.

35. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, and HLA-DR-.

36. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16- and CD66b-, HLA-DR-, CD123+.

37. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, and CD38+.

38. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19-, and CD3-.

39. The method of claim 32 or 33, wherein the cells expressing intracellular arginase 1 (ARG1+) are CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+, and CD38.

40. The method of any one of claims 1 to 39, wherein the immune checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand.

41. The method according to any one of claims 1 to 40, wherein The immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

42. The method of any one of claims 1 to 41, wherein the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

43. The method of any one of claims 1 to 42, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplizumab, durvalumab, atezolizumab, or avelumab.

44. The method of any one of claims 1 to 43, wherein the agent targeted to cells expressing arginase 1 targets cells constitutively expressing arginase 1.

45. A pharmaceutical composition comprising an agent that inhibits arginase 1 activity or targets cells expressing arginase 1 and an immune checkpoint inhibitor.

46. A kit for treating cancer, comprising: (a) an agent that inhibits arginase 1 activity or targets cells expressing arginase 1; and optionally (b) Immune checkpoint inhibitors.

47. A method of determining whether a subject suffering from cancer will not respond to treatment with an immune checkpoint inhibitor, the method comprising: (i) providing positive response controls from one or more positive responders who have developed a clinical response to the treatment, and negative response controls from one or more non-responders who have not developed a clinical response to the treatment, each response control representing the distribution of cells from a blood sample of the one or more positive responders and the one or more non-responders within a series of categories, wherein the distribution of cells is based on the expression of a set of biomarkers by the cells of the sample, such that cells with the same biomarker expression of the set of biomarkers are classified into the same category, wherein the set of biomarkers includes intracellular arginase 1 and at least four markers selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD1 4, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD123, CD127, CD25, CD33, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, CD274 (PD-1), CD279 (PD-L1), TIGIT, CD38, Ki67, granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and GM-CSFRα chain; (ii) determining the distribution of cells in a blood sample from the subject within the series of categories of the positive-responding control and the negative-responding control; (iii) comparing the distribution of the cells from the sample from the subject within the series of categories with the negative response control and / or the positive response control, wherein if the distribution of the cells from the sample from the subject within the series of categories is substantially the same as the distribution of the cells represented by the negative response control within the series of categories and / or different from the distribution of the cells represented by the positive response control within the series of categories, the subject is determined to be non-responsive to the treatment.

48. The method of claim 47, wherein the panel of biomarkers comprises intracellular arginase 1 and at least 8 markers selected from the group consisting of CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD66b, CD304, CD123, CD127, CD25, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), CD196 (CCR6), CD197 (CCR7), integrin beta 7, and GM-CSFR alpha chain.

49. The method of claim 47, wherein the panel of biomarkers comprises intracellular arginase 1 and at least 13 markers selected from the group consisting of: CD45, CD3, CD20, CD14, CD15, CD16, CD11b, CD19, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), and GM-CSFR alpha chain.

50. The method of claim 47, wherein the panel of biomarkers includes intracellular arginase 1 and CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, and CD56.

51. The method of claim 47, wherein the panel of biomarkers includes intracellular arginase 1 and CD3, HLA-DR, CD14, CD16, CD19, CD66b, CD45RO, and CD38.

52. The method of claim 47, wherein the panel of biomarkers includes intracellular arginase 1 and CD3, CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, and CD56.

53. The method of any one of claims 47 to 52, wherein (ii) determining the distribution of cells of the sample within the series of classes comprises the steps of: a. assessing the expression of each cell of a sample from the subject for each biomarker in the set of biomarkers in response to the control; b. classifying each cell of the sample from the subject into one of the series of categories of the response control; c. measuring the number of cells of the test sample in each category of the series of categories in the response control; The distribution of cells from the sample of the subject within the series of categories of the response control is thereby determined.

54. The method of claim 53, wherein (iii) comparing the distribution of cells from the sample from the subject within the series of categories comprises the steps of: comparing the measured number of cells in each category of the series of categories of the response control from the sample from the subject to the number of cells in each category of the series of categories of the response control; It is thereby determined whether the distribution of cells of the sample within the series of categories of the response controls is the same as the distribution of cells represented by one of the response controls.

55. The method of any one of claims 47 to 54, wherein the serial categories include the following categories: CD8+ Naive % Total, CD8+ % Total, CD8+CD45RO- Naive % Total, B Cells % Total, CD3+ % Total, CD4+ % Total, CD4 Naive % Total, CD4 Naive % CD4, CD4+TCM % Total, Treg % Total, Treg CD45RO+ % Total, IntB7 % RO, CD8+CD45RO+ Memory % CD8, NK Cells % CD3-CD20-, CCR6 % RO, Monocytes % Total, CD14 Monocytes % Total, and NK Cells % Total.

56. The method of any one of claims 32 to 39, wherein the sample is a peripheral blood sample.

57. The method of any one of claims 1 to 56, wherein the cancer is non-small cell lung cancer (NSCLC).

58. A method of determining whether a subject will not respond to treatment with an immune checkpoint inhibitor, comprising determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject; Wherein if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated relative to a positive responding control, then the subject is determined to be non-responsive to the treatment.

59. The method of claim 58, wherein the positive response control is the level of ARG1+, CD14-, CD16-, CD66b- cells in a blood sample from a subject or more than one subject that responds to the immune checkpoint inhibitor treatment.

60. The method of claim 58 or 59, wherein the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-.

61. The method of claim 58 or 59, wherein the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+.

62. The method of claim 58 or 59, wherein the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, CD38+.

63. The method of claim 58 or 59, wherein the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19-, CD3-.

64. The method of claim 58 or 59, wherein the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+CD38+.

65. The method of any one of claims 58 to 64, wherein the immune checkpoint inhibitor is an inhibitor of the interaction between PD-1 and its ligand.

66. The method according to any one of claims 58 to 65, wherein The immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

67. The method of any one of claims 58 to 66, wherein the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

68. The method of any one of claims 58 to 67, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplizumab, durvalumab, atezolizumab, or avelumab.

Images