Methods and Treatments

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to methods of treating cancer in a human subject who is unresponsive to treatment with an immune checkpoint inhibitor, methods of reducing the unresponsiveness of a subject to treatment with an immune checkpoint inhibitor, and compositions and kits for such uses.

[0002] background Immune checkpoint therapy is a form of cancer treatment that modulates a patient's immune system and treats 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 suppress the body's immune response to cancer cells.

[0003] Immune checkpoint inhibitors approved and / or in clinical trials for the treatment of cancer include the anti-PD-1 antibodies nivolumab (for metastatic melanoma) and pembrolizumab (for the treatment of metastatic melanoma, lymphoma, mesothelioma, and non-small cell lung cancer), and the anti-PD-L1 antibodies, including 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)).

[0004] However, a major problem with cancer treatment with immune checkpoint inhibitors is the high incidence of primary non-responders. For example, approximately 60% to 70% of metastatic melanoma patients treated with anti-PD-1 antibodies do not respond to treatment.

[0005] WO 2019 / 051542 describes a method for identifying subjects likely to respond to treatment with immune checkpoint inhibitors. While such an approach identifies and facilitates treatment of patients who are responsive to checkpoint inhibitors, treating cancer patients who are unresponsive to immune checkpoint inhibitors remains problematic.

[0006] There is a need for improved methods of treating cancer in patients who are unresponsive to immune checkpoint inhibitors.

[0007] overview We found that a subset of cells with elevated levels of intracellular arginase 1 plays an important role in the lack of responsiveness to immune checkpoint inhibitors that target the interaction between PD-1 and its ligands.

[0008] As described in the Examples, the inventors have found that non-responsiveness to immune checkpoint therapy targeting the interaction between PD-1 and its ligand is closely correlated with an elevated population of a specific subset of cells with elevated intracellular levels of arginase 1. Thus, the inventors predict that inhibiting arginase 1 activity in the subset of cells with elevated intracellular levels of arginase 1 can reverse a subject's non-responsiveness to treatment with an immune checkpoint inhibitor, thereby enabling the subject to subsequently be effectively treated with an immune checkpoint inhibitor.

[0009] Accordingly, a first aspect is a method of treating cancer in a subject who has been identified as non-responsive to treatment with an immune checkpoint inhibitor, comprising: (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to an immune checkpoint inhibitor; (b) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; wherein the subject (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, wherein the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD1 providing a marker comprising at least five markers selected from CD1c, 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-CSFR alpha chain, and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides a method for treating a leukemia, wherein the leukemia is identified as being non-responsive to immune checkpoint inhibitors by

[0010] A second aspect is a method of treating cancer in a human subject, comprising: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, wherein the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD1 providing a marker comprising at least five markers selected from CD1c, 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-CSFR alpha chain, and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor; (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; The present invention provides a method comprising:

[0011] A third aspect is a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by each cell of the sample, wherein cells having the same expression of the group of biomarkers are classified into the same class, and the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD1 1b, 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-CSFR α chain, and intracellular arginase 1. (ii) determining the distribution of cells of a sample from a subject within a series of classes of positive and negative reaction controls, wherein the sample is in the form of a sample of cells from the subject's blood; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to have a likelihood of being non-responsive to the treatment if the distribution of cells of the sample within the set of classes from the subject is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing the likelihood that the subject will be non-responsive to treatment with an immune checkpoint inhibitor, The method provides a method for determining whether a subject is non-responsive to an immune checkpoint inhibitor by:

[0012] In one embodiment, the group of biomarkers comprises at least 13 markers selected from 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 beta7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase.

[0013] In one embodiment, the group of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0014] In one embodiment, the group of biomarkers comprises at least 13 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 β7, GM-CSFR α chain, and intracellular arginase 1.

[0015] In one embodiment, the group of biomarkers comprises at least 13 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, and intracellular arginase 1.

[0016] In one embodiment, the group of biomarkers comprises CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56 and intracellular arginase 1.

[0017] In one embodiment, the group of biomarkers comprises HLA-DR, CD14, CD16, CD66b, CD45RO, CD38 and intracellular arginase 1.

[0018] In one embodiment, the group of biomarkers comprises HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38 and intracellular arginase 1.

[0019] In one embodiment, the group of biomarkers comprises HLA-DR, CD14, CD16, CD66b and intracellular arginase 1.

[0020] In one embodiment, (ii) determining the distribution of cells of the sample within a set of classes comprises: a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes to determine the distribution of cells of a sample from a subject within a series of classes of response controls.

[0021] In one embodiment, (iii) comparing the distribution of cells of the sample from the subject within a set of classes comprises: comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; to determine whether the distribution of cells of the sample within a set of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0022] In one embodiment, the set of classes includes naive Tcon in CD4+, CD4+ Treg in live cells, CD4+ Tcon in live cells, β7+ in CD45RO+ CD4+ Tcon, CCR6+ in CD45RO+ CD4+ Tcon, naive CD8+ in live cells, naive CD8+ in live cells, CD8+ (in) CD45RO+, CD16loCD56hi NK in live cells, CD14+CD16- classical monocyte in live cells, CD4+ in live cells, CD8+ in live cells, naive in CD8+.

[0023] In one embodiment, the set of classes includes the following classes: % CD8+ naive to total, % CD8+ to total, % CD8+CD45RO- naive to total, % B cells to total, % CD3+ to total, % CD4+ to total, % CD4 naive to total, % CD4 naive to CD4, % CD4+ TCM to total, % Treg to total, % Treg CD45RO+ to total, % IntB7 to RO, % CD8+CD45RO+ memory to CD8, % NK cells to CD3-CD20-, % CCR6 to RO, % monocytes to total, % CD14 monocytes to total, and % NK cells to total. In one embodiment, the sample is a peripheral blood sample.

[0024] In one embodiment, the method further includes monitoring the effectiveness of the treatment, comprising 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.

[0025] In one embodiment, the group of biomarkers comprises 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 13 markers selected from CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin β7.

[0026] A fourth aspect provides a method of treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, and administering the effective amount of the immune checkpoint inhibitor, optionally in combination with further administration of the agent that inhibits arginase 1 activity and / or the agent that targets cells that express arginase 1.

[0027] A fifth aspect is a method of treating cancer in a human subject, comprising: (a) assessing whether a subject is non-responsive to treatment with an immune checkpoint inhibitor, by measuring intracellular arginase 1 (ARG1+) and CD14 expression in a blood sample from the subject; - , CD16 - , CD66b - and determining the level of cells that express intracellular arginase 1 (ARG1+) and CD14 in the sample relative to a positive control. - , CD16 - , CD66b - assessing whether the subject is non-responsive to treatment if the level of cells that are (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; The present invention provides a method comprising:

[0028] A sixth aspect is a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: 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 treatment if the level of cells expressing intracellular arginase 1 (ARG1+) and which are CD14-, CD16-, and CD66b- in the sample is elevated compared to a positive response control; The method provides a method for determining whether a subject is non-responsive to an immune checkpoint inhibitor by:

[0029] In one embodiment, cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16- and CD66b-.

[0030] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b- and HLA-DR-.

[0031] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16- and CD66b-, HLA-DR-, and CD123+.

[0032] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+ and CD38+.

[0033] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19- and CD3-.

[0034] In one embodiment, the cells expressing intracellular arginase 1 (ARG1+) are CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+ and CD38+.

[0035] In one embodiment of the fifth or sixth aspect, the positive response control is the level of cells that express intracellular arginase 1 (ARG1+) and are CD14-, CD16-, and CD66b- in a blood sample from a subject or subjects who are responsive to treatment with an immune checkpoint inhibitor.

[0036] In one embodiment, the immune checkpoint inhibitor is an inhibitor of the interaction of PD-1 with its ligand.

[0037] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0038] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

[0039] In one embodiment, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, durvalumab, atezolizumab, or avelumab.

[0040] In one embodiment, the agent that targets cells that express Arginase 1 targets cells that constitutively express Arginase 1.

[0041] A seventh aspect provides a pharmaceutical composition comprising an agent that inhibits Arginase 1 activity or targets cells that express Arginase 1, and an immune checkpoint inhibitor.

[0042] An eighth aspect is a kit for treating cancer, comprising: (a) an agent that inhibits arginase 1 activity or targets cells that express arginase 1; and, optionally, (b) Immune checkpoint inhibitors and A kit is provided, comprising:

[0043] A ninth aspect is a method of assessing whether a subject suffering from cancer is non-responsive to treatment with an immune checkpoint inhibitor, comprising: (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, each response control representing a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by cells of the sample, cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being intracellular arginase 1, as well as CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, C providing a marker comprising at least four markers selected from D16, 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-CSFR α chain; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides a method comprising:

[0044] In one embodiment, the group of biomarkers comprises intracellular arginase 1 and at least eight markers selected from 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.

[0045] In one embodiment, the group of biomarkers comprises intracellular arginase 1 and at least 13 markers selected from CD45, CD3, CD20, CD14, CD15, CD16, CD11b, CD19, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), and GM-CSFR alpha chain.

[0046] In one embodiment, the group of biomarkers comprises intracellular arginase 1, as well as CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3 and CD56.

[0047] In one embodiment, the group of biomarkers comprises intracellular arginase 1, as well as CD3, HLA-DR, CD14, CD16, CD19, CD66b, CD45RO and CD38.

[0048] In one embodiment, the group of biomarkers comprises intracellular arginase 1, as well as CD3, CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19 and CD56.

[0049] In one embodiment, (ii) determining the distribution of cells of the sample within a set of classes comprises: a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes to determine the distribution of cells of a sample from a subject within a series of classes of response controls.

[0050] In one embodiment, (iii) comparing the distribution of cells of a sample from a subject within a set of classes comprises: comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; to determine whether the distribution of cells of the sample within a set of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0051] In one embodiment, the set of classes includes % CD8+ naive to total, % CD8+ to total, % CD8+CD45RO- naive to total, % B cells to total, % CD3+ to total, % CD4+ to total, % CD4 naive to total, % CD4 naive to CD4, % CD4+ TCM to total, % Treg to total, % Treg CD45RO+ to total, % IntB7 to RO, % CD8+CD45RO+ memory to CD8, % NK cells to CD3-CD20-, % CCR6 to RO, % monocytes to total, % CD14 monocytes to total, and % NK cells to total classes.

[0052] A tenth aspect provides a method for determining whether a subject is non-responsive to treatment with an immune checkpoint inhibitor, the method comprising determining the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from the subject, wherein the subject is determined to be non-responsive to treatment if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated compared to a positive responder control.

[0053] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-.

[0054] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+.

[0055] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, CD38+.

[0056] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD45RO+, CD38+, CD19-, CD3-.

[0057] In one embodiment, the ARG1+, CD14-, CD16-, CD66b- cells are ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD45RO+, CD38+.

[0058] In one embodiment, the immune checkpoint inhibitor is an inhibitor of the interaction of PD-1 with its ligand.

[0059] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0060] In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody or an anti-PD-L1 monoclonal antibody.

[0061] In one embodiment, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, durvalumab, atezolizumab, or avelumab.

[0062] In one embodiment, the blood sample is a peripheral blood sample.

[0063] In one embodiment, the cancer is non-small cell lung cancer (NSCLC) or colon cancer.

[0064] An alternative first aspect is an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, or the use of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 in the manufacture of a medicament for treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, wherein the subject: (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, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP 3. providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers, selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from a subject within a series of classes of positive and / or negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides an agent and an immune checkpoint inhibitor, or a use of an agent, which has been identified as being non-responsive to immune checkpoint inhibitors by

[0065] An alternative second embodiment is an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, or the use of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 in the manufacture of a medicament for treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, wherein the subject: (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and / or negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, wherein the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, F providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers selected from oxP3, 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from a subject within a series of classes of positive and / or negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides an agent and an immune checkpoint inhibitor, or a use of an agent, which has been identified as being non-responsive to immune checkpoint inhibitors by

[0066] An alternative third aspect is an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 for use in increasing responsiveness to immune checkpoint inhibitor treatment in a subject suffering from a treatment-nonresponsive cancer, or the use of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1 in the manufacture of a medicament for increasing responsiveness to immune checkpoint inhibitor treatment in a subject suffering from a treatment-nonresponsive cancer, wherein the subject (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, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP 3. providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers, selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from a subject within a series of classes of positive and / or negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides an agent or use of an agent that has been identified as being non-responsive to immune checkpoint inhibitors by

[0067] An alternative fourth aspect provides an agent that inhibits arginase 1 activity or targets cells that express arginase 1 and an immune checkpoint inhibitor, or use of an agent that inhibits arginase 1 activity or targets cells that express arginase 1, in combination with an immune checkpoint inhibitor, for use in treating cancer in a subject who is non-responsive to treatment with an immune checkpoint inhibitor, wherein the subject is non-responsive to treatment with an immune checkpoint inhibitor, or use of the agent and immune checkpoint inhibitor, or agent, in the manufacture of a medicament for treating cancer in a subject.

[0068] An alternative fifth embodiment relates to an agent that inhibits Arginase 1 activity or targets cells that express Arginase 1 and an immune checkpoint inhibitor for use in treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, or the use of an agent that inhibits Arginase 1 activity or an agent that targets cells that express Arginase 1 in combination with an immune checkpoint inhibitor in the manufacture of a medicament for treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, wherein the subject is (a) determining the level of cells that express intracellular arginase 1 (ARG1+) and are CD14-, CD16-, CD66b- in a blood sample from the subject relative to a positive responder control, wherein the subject is determined to be non-responsive to treatment if the level of cells that express intracellular arginase 1 (ARG1+) and are CD14-, CD16-, CD66b- in the sample is elevated relative to the positive responder control; The present invention provides an agent and an immune checkpoint inhibitor, or a use of an agent, which has been identified as being non-responsive to an immune checkpoint inhibitor by a method comprising: [Brief explanation of the drawings]

[0069] [Figure 1]This heatmap shows pretreatment blood immune signature populations in 10 NSCLC patients and one healthy control. Data from 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) were analyzed by MeV. Columns represent samples from each individual. Rows represent the measured parameters (% of cells in each subpopulation; rows are normalized to show SD from the mean, as indicated by the top bar; black indicates at least 1.5 SD below the mean, and light gray indicates at least 2 SD above the mean). Five patients failed to generate a clinical response to anti-PD-L1 therapy (non-responders, indicated by black bars). Five patients developed a clinical response to anti-PD-L1 therapy (responders, indicated by green bars). Hierarchical clustering using Pearson correlation with average linkage showed that data from the five non-responder patients clustered similarly to data from the five responder patients, demonstrating the utility of the 13-mAb panel. [Figure 2] Dot plot from mass cytometry analysis of a non-responder NSCLC patient. Arginase 1-expressing cells (0.37% of the total) are gated (black polygon) and clearly distinct from the remaining cells, which express lineage markers (CD3, CD14, CD16, CD66b, and CD19). Arginase 1-expressing cells are HLA-DRlo, CD38hi, and CD45RO+. [Figure 3] Graph showing the percentage of intracellular arginase 1-expressing cells as a function of clinical response to anti-PD-1 / PD-L1 therapy in NSCLC. **p<0.01 [Figure 4] Graphs showing the lack of correlation between plasma arginase or arginine and clinical response to anti-PD-1 / PD-L1 therapy in NSCLC. Left panel: plasma arginase activity. Right panel: plasma l-arginine. ns: not significant. [Figure 5]1 is a graph showing the correlation between plasma arginase activity and plasma arginine in NSCLC.

[0070] Detailed Description A major problem with cancer treatment with immune checkpoint inhibitors is that many patients do not respond to treatment during the initial treatment period, and these patients are referred to as primary non-responders. The present inventors have demonstrated that in non-responders, the main cell populations that decrease are CD4 T cells (specifically, regulatory T cells (Treg), naive conventional (i.e., non-regulatory) CD4 T cells (abbreviated here as Tconv or Tcon), and central memory CD4 Tconv naive CD8 T cells) and NK cells, and not classical monocytes and HLA-DR1. + Conversely, we have previously found an increase in naive CD4 and CD8 T cells (monocytes and B cells) in non-responders. Without wishing to be bound by theory, we believe that the decrease in naive CD4 and CD8 T cells in non-responders is due to a systemic effect, since naive T cells circulate in the blood and secondary lymphoid tissues such as lymph nodes (LNs) and the spleen, but are generally excluded from peripheral non-immune tissues such as tumors.

[0071] The number of naive T cells is regulated by thymic output and peripheral lifespan. Given the advanced age of our cohort and the low thymic output in elderly subjects, we believe that a decrease in peripheral lifespan (i.e., reduced survival) is likely the direct cause of the decline in naive CD4 and CD8 T cells. One of the most important requirements for the survival of naive T cells is low levels of T cell receptor (TCR) signaling via dendritic cells in secondary lymphoid tissues. Peripheral T cells with acutely suppressed TCR expression rapidly die. Recognition of self-antigens in secondary lymphoid organs results in partial phosphorylation, which extends the survival of naive T cells.

[0072] The inventors believe that reduced TCR signaling is predicted to reduce both T cell survival and T cell responsiveness to antigen. The inventors believe that the inability of tumor-sensitized cells to mount an effective attack against tumors after administration of an agent that blocks the PD-1 / PD-L1 pathway is due to reduced T cell responsiveness to antigen, and that reduced T cell survival accounts for the concomitant loss of multiple T cell subsets.

[0073] 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.

[0074] Recent immunological research in cancer has focused on the pre-treatment tumor microenvironment, where tumor-specific immune cells are highly enriched compared to the circulation. In contrast, it has been reported that the T cells responsible for tumor clearance in response to anti-PD-1 therapy have a different TCR repertoire than the "exhausted" cells in the tumor microenvironment before checkpoint therapy. Therefore, we believe that the T cells responsible for tumor clearance in response to anti-PD-1 therapy must react to anti-PD-1 somewhere other than the tumor (the most likely site is the tumor-draining lymph node) and then migrate into the tumor in response to therapy.

[0075] As described in the Examples, the inventors analyzed peripheral blood mononuclear cells from a cohort of non-small cell lung cancer patients before and after treatment with anti-PD-1 / PD-L1 immune checkpoint inhibitors. The inventors found a statistically significant negative correlation between the number of intracellular arginase 1-expressing cells in the blood and clinical response. The inventors also found that the correlation between clinical response and plasma arginase and arginine was not statistically significant (although, as expected, plasma arginine activity was higher in samples with lower arginine content). This indicates that cells with high intracellular arginase 1 are a major driver of downregulation of T cell responses, thereby impairing T cell responses to immune checkpoint inhibitors to the extent that they are unable to produce an effective clinical response.

[0076] Therefore, we propose that agents targeting arginase 1 be used to increase responsiveness in subjects with cancers that are unresponsive to immune checkpoint inhibitors, potentially reverting the unresponsive immune signature to normal over time.

[0077] Accordingly, one aspect is a method of treating cancer in a human subject, comprising: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor; (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; The present invention provides a method comprising:

[0078] In one aspect, there is provided a method of treating cancer in a human subject, comprising: (a) (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, wherein each response control represents a distribution of cells of a blood sample from the positive responders or non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, wherein the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP 3. providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers, selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from a subject within a series of classes of positive and / or negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a positive response control and / or a negative response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor; (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; A method is provided, comprising:

[0079] Another aspect is a method of treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, comprising: (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to an immune checkpoint inhibitor; (b) then administering an effective amount of an immune checkpoint inhibitor, optionally in combination with a further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; wherein the subject (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides a method for treating a leukemia, wherein the leukemia is identified as being non-responsive to immune checkpoint inhibitors by

[0080] Another aspect is a method of treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, comprising: (a) administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to an immune checkpoint inhibitor; (b) then administering an effective amount of an immune checkpoint inhibitor, optionally in combination with a further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; wherein the subject (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, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, the group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD11b, CD11c, CD66b, CD304, FoxP 3. providing at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers, selected from 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 beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in a blood sample from a subject within a series of classes of positive and / or negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; The present invention provides a method for treating a leukemia, wherein the leukemia is identified as being non-responsive to immune checkpoint inhibitors by

[0081] In one embodiment, (ii) determining the distribution of cells of the sample within a set of classes comprises: a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes to determine the distribution of cells of a sample from a subject within a series of classes of response controls.

[0082] In one embodiment, (iii) comparing the distribution of cells of the sample from the subject within a set of classes comprises: comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; to determine whether the distribution of cells of the sample within a set of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0083] Type of clinical response The first step of the method involves assessing whether a subject with a cancer known to be responsive to treatment with an immune checkpoint inhibitor is likely to be responsive or non-responsive to treatment with an immune checkpoint inhibitor.

[0084] A subject is responsive to treatment with an immune checkpoint inhibitor if they exhibit a complete clinical response, a partial clinical response, or stable disease.

[0085] A subject is non-responsive to treatment with an immune checkpoint inhibitor if they exhibit progressive disease.

[0086] As is commonly understood, a "complete response" to a therapy is generally understood to mean that all detectable signs of cancer disappear in response to treatment. A "partial response" is generally understood to mean a reduction in the tumor burden in an individual, e.g., in terms of the number, size, and growth rate of tumors. A partial response may extend the time until 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.

[0087] A complete clinical response, partial clinical response, stable disease, or progressive disease is, in one embodiment, one of the following: RECIST 1.0 criteria (Therasse P, et al.) 2000 J. Natl Cancer Inst 92:2015-16 can be determined by

[0088] RECIST 1.0 criteria A. Definition of Measurable and Nonmeasurable Disease Measurable disease: Presence of at least one measurable lesion.

[0089] Measurable lesions: Longest diameter (LD) - 20 mm or more by conventional techniques (medical photography [skin or oral lesions], palpation, plain X-ray, CT or MRI) or Spiral CT scan: 10mm or more A lesion in which at least one dimension can be accurately measured.

[0090] Non-measurable lesions: All other lesions, including bone lesions, leptomeningeal disease, ascites, pleural or pericardial effusion, cutaneous / pulmonary lymphangitis, abdominal masses not identified and followed by imaging techniques, cystic lesions, or lesions that are too small to be considered measurable (longest diameter less than 20 mm by conventional techniques or less than 10 mm by spiral CT scan), including disease reported only by indirect evidence (e.g., laboratory values).

[0091] B.Measurement method 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 continuously at a minimum of 10 mm. MRI is preferred; if used, subsequent examinations should measure the lesion in the same anatomical plane using the same imaging sequence. Whenever possible, the same scanner should be used.

[0092] Spiral CT: The minimum baseline lesion size can be 10 mm, provided that images are serially reconstructed at 5 mm intervals. This specification applies to tumors of the chest, abdomen, and pelvis.

[0093] Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions if they are well-defined and surrounded by aerated lung. However, MRI is preferred.

[0094] Clinical Examination: Clinically detected lesions are considered measurable by RECIST only if they are superficial (e.g., skin nodules and palpable lymph nodes). Skin lesions require documentation by color photographs, including a ruler in the field of view to estimate the size of the lesion, and the patient study number.

[0095] C. Baseline recording of target and non-target lesions All measurable lesions, up to a maximum of 5 lesions per organ and a total of 10 lesions, representing all affected organs, should be identified as target lesions and recorded and measured at baseline.

[0096] Target lesions should be selected based on their size (lesions with LD) and their amenability to accurate repeated measurement (clinical or by imaging techniques). The sum of the LD of all target lesions is calculated and reported as the baseline total LD. The baseline total LD ​​is used as the criterion for characterizing objective tumor response.

[0097] All other lesions (or disease sites) should be identified as non-target lesions and should also be recorded at baseline. Measurement of these lesions is not required, but their presence or absence should be recorded throughout follow-up.

[0098] Records of index lesions should include the date of assessment, description of the lesion location, dimensions, and type of diagnostic test used to follow the lesion.

[0099] All measurements should preferably be taken and recorded in metric units using a ruler or calipers.

[0100] D. Reaction Criteria Disease assessments will be performed every 6 weeks after initiation of treatment. However, subjects who experience a partial or complete response should undergo a confirmatory disease assessment at least 28 days later. The assessment should occur as close to, but preferably after, 28 days (as scheduling permits).

[0101] The definitions for the target lesion response ratings are as follows: Target Lesion Evaluation Complete response (CR) - disappearance of all target lesions. Partial response (PR) - at least a 30% reduction in the sum of the LD of target lesions relative to the baseline total LD. Stable disease (SD) - Neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as progressive disease (PD), based on the smallest total LD ​​recorded since treatment initiation. Lesion or appearance of one or more new lesions, based on the smallest total LD ​​recorded since treatment initiation.

[0102] E. Evaluation of Non-Target Lesions The criteria used to determine objective tumor response for non-target lesions are defined as follows: Complete response - disappearance of all non-target lesions. Incomplete response / stable disease - persistence of one or more non-target lesions. Progressive disease - Appearance of one or more new lesions and / or overt progression of existing non-target lesions.

[0103] F. Evaluation of overall response based on RECIST criteria Overall response is the best recorded response from the start of treatment until confirmed disease progression / recurrence. Generally, a subject's best response assignment depends on the achievement of both the measurement and confirmation criteria.

[0104] The table below shows the best overall response assessment for all possible combinations of tumor response in target and non-target lesions, with or without the appearance of new lesions.

[0105] [Table 1]

[0106] Note: Subjects who experience a global deterioration in health status requiring discontinuation of treatment but who do not have objective evidence of disease progression at that time should be classified as having "symptomatic deterioration." Every effort should be made to document objective progression, even after treatment has been discontinued.

[0107] In some situations, it may be difficult to distinguish between residual disease and normal tissue. If assessment of complete response depends on this determination, it is recommended that residual disease be investigated (fine needle aspiration / biopsy) to confirm complete response status.

[0108] G. Confirmation Criteria To be assigned a PR or CR status, confirmatory disease assessment should occur at least 28 days after first meeting response criteria.

[0109] To be assigned to SD status, at least one follow-up measurement, spaced a minimum of 12 weeks apart after study enrollment, had to meet SD criteria.

[0110] Preparation of reaction controls The methods disclosed herein may further include assessing one or more tissues or organs of an individual administered an immune checkpoint inhibitor to determine tumor regression in the individual and, therefore, whether the individual is suitable for providing a positive or negative response control. In one embodiment, this step utilizes radiological imaging to determine the location and volume of each of multiple tumor lesions in the subject after treatment with an immune checkpoint inhibitor. For example, this may include three-dimensional radiological images of the subject that register the geographic location of each of multiple tumor lesions. Non-limiting examples of radiological images that can be used to determine the location 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.

[0111] The positive and / or negative response controls may be derived from a single individual. However, in some embodiments, the positive response control is preferably derived from a cohort or multiple positive responders. In some embodiments, the negative response control is derived from multiple non-responders.

[0112] As described herein, when a cohort or multiple positive responders or non-responders are used to derive a response control, the control is based on distribution data resulting from the individual evaluation of each positive responder or non-responder sample selected for the derivation of the positive response control or negative response control. Thus, each sample is evaluated to determine a cell distribution profile specific to each sample. The cell distribution profiles of the responders are then aggregated to form a positive response control, and the cell distribution profiles of the non-responders are aggregated to form a negative response control.

[0113] Aggregation may allow for the identification of cell populations primarily associated with lack of clinical response to therapy in a large proportion of non-responders. Relevant statistical methods will be understood by those skilled in the art and are further described herein.

[0114] In one embodiment, the positive response control represents the distribution of cells in a sample of a positive responder before or after treatment of the positive responder with an immunomodulatory agent. Preferably, the positive response control represents the distribution of cells in a sample of a positive responder before subjecting the positive responder to treatment with an immunomodulatory agent that elicits a positive response. In one embodiment, the negative response control represents the distribution of cells in a sample of a non-responder before or after therapy of the non-responder with an immune checkpoint inhibitor. Preferably, the negative response control represents the distribution of cells in a non-responder before subjecting the non-responder to treatment with an immune checkpoint inhibitor that elicits a negative response.

[0115] As described herein, the purpose of the positive and negative response controls is to provide a reference point from which a prediction can be made of the likelihood that another individual will not develop a clinical response to treatment with the same immune checkpoint inhibitor (i.e., the same as that used to generate the positive and negative response controls), preferably for the same cancer (i.e., the same as that treated in the individual from which the positive and negative response controls were derived). The prediction is made based on a comparison of the test sample (a sample from the subject) with the positive and / or negative response controls. The comparison can be a comparison of the frequency of cells in a defined class or subpopulation of cells between the positive and / or negative response controls and the test sample.

[0116] In one embodiment, the reaction control comprises the following steps: (i) providing a responder cell sample in the form of cells from an individual who has responded to therapy with an immune checkpoint inhibitor; (ii) providing a non-responder cell sample in the form of cells from an individual who has not responded to therapy with an immune checkpoint inhibitor; (iii) applying a cell distribution analysis to the responder cell sample to partition the cells in the responder cell sample into a series of classes according to the expression of a group of biomarkers by each cell, such that cells having the same expression of each biomarker of the group are grouped into the same class, thereby forming a responder cell distribution profile; (iv) applying the same cell distribution analysis to a sample of non-responder cells, thereby forming a non-responder cell distribution profile; (v) identifying the distribution of cells in the responder cell distribution profile that are not seen in the non-responder cell distribution profile, and / or the distribution of cells in the non-responder cell distribution profile that are not seen in the responder cell distribution profile; and a distribution of cells in the responder cell distribution profile that are not seen in the non-responder cell distribution profile is identified as a positive response control, and / or a distribution of cells in the non-responder cell distribution profile that are not seen in the responder cell distribution profile is identified as a negative response control.

[0117] Typically, the responder cell distribution profile is an aggregation of the cell distribution profiles of different responders, which increases the likelihood that the control will contain a cell distribution associated with response to therapy in a large proportion of individuals who responded or did not respond to therapy.

[0118] Typically, the multiple non-responder cell samples may be an aggregation of different non-responder cell distribution profiles.

[0119] Cells may be differentiated according to expression of at least 5, 6, 7, 8, 9, 10, 11, 12 or at least 13 biomarkers, although the number of biomarkers may in some embodiments be 20, 30, 40, 100 or more.

[0120] The expression "same expression" and its grammatical variations in the context of "cells with the same expression" are generally understood to refer to the level of identity or similarity between related subjects. The level of identity or similarity may be a function of the number of responder cell distribution profiles forming the positive response control or the number of non-responder cell distribution profiles forming the negative response control. When the response control is generated from an aggregation of responder or non-responder cell distribution profiles, it can be expected that "same expression" means a lower level of identity between related subjects than would be expected if the response control were obtained from a single responder or non-responder cell distribution profile.

[0121] Biomarkers In some embodiments, cells can be assessed for the absence of biomarker expression or the presence of biomarker expression. In some embodiments, cells can be assessed for a particular level of biomarker expression. For example, cells can be assessed for a "low" level of biomarker expression (e.g., CD4 lo ) or "high" levels of biomarker expression (e.g., CD4 hi The meaning of the level of biomarker expression designated as "hi" or "lo" is generally well understood by those skilled in the art. For example, CD4 hi and CD4 lo T cells or CD127 hi and CD127 lo The meaning of T cells is understood by those skilled in the art and is routinely determined using standard techniques.

[0122] For purposes of illustrating the method, in one example, cells are analyzed and the cells are identified as class CD25 + CD127 lo T reg In this example, CD25, CD125, and various other T reg Antibodies specific to the marker are available. In this example, CD25 + CD127 lo T reg Cells with CD25 are classified into this class. - CD127 lo T reg or CD25 + CD127 hi T reg or CD25 - CD127 hi T reg Cells with CD25 + CD127 lo T reg Not divided into classes.

[0123] Typically, biomarkers are molecules characteristically expressed by cells with immune function, including lymphocytes, monocytes, granulocytes, etc. In one embodiment, the group of biomarkers includes CD4 or CD8 T cell markers and 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, Glanza and at least 13 markers, typically at least 14, 15, 16, 17 or 18 markers from the group consisting of immunoglobulin B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0124] In one embodiment, the group of biomarkers comprises a CD4 or CD8 T cell marker and a combination 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, CD and at least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0125] In one embodiment, the group of biomarkers comprises a CD4 or CD8 T cell marker and a combination 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, CD 134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1, and typically at least 30, 31, 32, 33, 34, 35, 36, 37 or 38 markers from the group consisting of: CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0126] In one embodiment, the group of biomarkers is CD4 or CD8 The present invention relates to a method for detecting T cell markers and at least 39 markers selected from the group consisting of 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 β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0127] In one embodiment, the panel of biomarkers comprises molecules expressed by B cells or plasma cells.

[0128] In one embodiment, the group of biomarkers comprises a B cell or plasma cell marker and 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, C and at least 13 markers, typically at least 14, 15, 16, 17 or 18 markers from the group consisting of D38, 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.

[0129] In one embodiment, the group of biomarkers comprises a B cell or plasma cell marker and 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, Ki 67, Granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1, and typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of:

[0130] In one embodiment, the group of biomarkers comprises a CD4 or CD8 T cell marker and a combination 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, CD 134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1, and typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of: CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0131] In one embodiment, the group of biomarkers comprises a B cell or plasma cell marker and 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 and at least 39 markers selected from the group consisting of (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.

[0132] In one embodiment, the group of biomarkers comprises molecules expressed by NK cells.

[0133] In one embodiment, the group of biomarkers comprises NK cell markers and 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, and typically at least 14, 15, 16, 17 or 18 markers from the group consisting of: CD40, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0134] In one embodiment, the group of biomarkers comprises NK cell markers and 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, At least 19 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of Granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0135] In one embodiment, the group of biomarkers comprises NK cell markers and 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, At least 29 markers, typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of Granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0136] In one embodiment, the group of biomarkers comprises NK cell markers and 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 and at least 39 markers selected from the group consisting of: CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0137] In one embodiment, the panel of biomarkers comprises molecules expressed by monocytes or dendritic cells.

[0138] In one embodiment, the group of biomarkers comprises a monocyte or dendritic cell marker and 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, CD 38, 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, and typically at least 13 markers, typically at least 14, 15, 16, 17, or 18 markers.

[0139] In one embodiment, the group of biomarkers comprises a monocyte or dendritic cell marker and 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, Ki6 7, Granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1, and typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of: CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0140] In one embodiment, the group of biomarkers comprises a monocyte or dendritic cell marker and 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, Ki6 7, Granzyme B, CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1, and typically at least 20, 21, 22, 23, 24, 25, 26, 27 or 28 markers from the group consisting of: CD134 (OX40), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin β7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10 and intracellular arginase 1.

[0141] In one embodiment, the group of biomarkers comprises a monocyte or dendritic cell marker and 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 and at least 39 markers selected from the group consisting of (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.

[0142] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0143] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7), and integrin β7. In one embodiment, using this group of biomarkers, the set of classes is: CD45RO+CD8+; CD14+, CD16-, classical monocytes in live cells; CCR6+ in CD45RO+, CD4+Tcon; CD4+ in live cells; CD4+Tcon in live cells; CD4+Treg in live cells; CD8+ in live cells; β7+ in CD45RO+CD4+Tcon; naive CD8+ in live cells; naive Tcon in CD4+; naive in CD8+; and CD16 in live cells. lo CD56 hi , NK.

[0144] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of at least 13 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 β7, GM-CSFR α chain, and intracellular arginase 1.

[0145] In one embodiment, the group 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 β7, and GM-CSFR α chain.

[0146] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of at least 13 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, and intracellular arginase 1.

[0147] In one embodiment, the group 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.

[0148] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

[0149] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, CD56, and intracellular arginase 1.

[0150] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD14, CD16, CD66b, CD45RO, CD38 and intracellular arginase 1.

[0151] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD3, CD14, CD16, CD19, CD66b, CD45RO, CD38 and intracellular arginase 1.

[0152] In one embodiment, the group of biomarkers comprises, consists essentially of, or consists of HLA-DR, CD14, CD16, CD66b, and intracellular arginase 1.

[0153] In one embodiment, the positive response control can represent the distribution of cells within a set of cells of at least 5, 10, 20, 30, 40, 50, 100, 150 or 200 classes.

[0154] In one embodiment, the negative reaction control can represent the distribution of cells within a set of cells of at least 5, 10, 20, 30, 40, 50, 100, 150 or 200 classes.

[0155] In one embodiment, the cells of the test sample are not assessed for T cell or B cell receptor diversity.

[0156] A positive response control can be expressed as the distribution of positive responder cells as the number of cells per class as a % of the total number of cells. A negative response control can be expressed as the distribution of non-responder cells as the number of cells per class as a % of the total number of cells. In one embodiment, when the checkpoint inhibitor is an anti-PD-1 antibody, the distribution of cells assessed is at least 5, 6, 7, 8, 9, or at least 10 classes of cells according to Table 1 below.

[0157] In one embodiment, when the checkpoint inhibitor is an anti-PD-1 antibody, the distribution of cells assessed is ARG1+ and at least 4, 5, 6, 7, 8 or at least 9 classes of cells according to Table 1 below.

[0158] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0159] The phrase "substantially the same as the distribution" and its grammatical variations in the context of "determining whether the distribution of cells of the test sample within a series of classes of negative reaction controls is the same as the distribution of cells represented by the negative reaction controls" is generally understood to refer to the level of similarity or identity between the test sample and the negative reaction controls. Preferably, the distribution is the same across at least most classes of negative reaction controls. In some embodiments, there may be some differences in the distribution across a minority of classes of negative reaction controls.

[0160] In one embodiment, the responder and non-responder cells are obtained from peripheral blood. The cells may be enriched for leukocyte subsets. For example, the cells may be enriched for mononuclear or polynuclear cells.

[0161] Assessment of positive responder and / or non-responder cells selected for formation of reaction controls can be performed by mass cytometry methods, including time-of-flight cytometry, as described in WO 2019 / 051542, following the methodology described below for assessment of cells in test samples.

[0162] Determining the distribution of cells in a test sample (i.e., a sample from a subject) The distribution of cells in the test sample can be determined by the following steps: a. assessing each cell of the test sample for expression of each biomarker in a panel of positive and / or negative response control biomarkers; b. classifying each cell of the test sample into one of a series of classes of positive and / or negative control; c. Measuring the number of cells in the test sample in each class of a series of positive and / or negative control classes The determination can be made in accordance with the following.

[0163] Assessment of the distribution of cells in a test sample involves considering the relative number of cells within each class of a series of classes in a positive and / or negative reaction control. This can also be expressed as the number of cells per class as a percentage of the total number of cells in the test sample. These assessments can be accomplished by mass cytometry methods, including time-of-flight cytometry, which allows assessment of individual cells for the expression of multiple biomarkers (40 or more) per cell. [Spitzer MH and GP Nolan 2016 Cell 165: 780-791] Please refer to.

[0164] Comparison of test samples with reaction controls Determining whether the distribution of cells of the test sample within a series of classes of the negative reaction control is the same as the distribution of cells represented by the negative reaction control generally involves: a. Comparing the measured cell count of the test sample in each class of the series of classes of negative reaction controls with the cell count of the negative reaction control in each class of the series of classes of negative reaction controls. requires.

[0165] Determining whether the distribution of cells of a test sample within a series of classes of a positive reaction control differs from the distribution of cells represented by the positive reaction control generally involves: b. Comparing the measured cell count of the test sample in each class of the series of classes of the positive reaction control with the cell count of the positive reaction control in each class of the series of classes of the positive reaction control. requires.

[0166] These steps can be performed in silico using available software packages.

[0167] In one embodiment, the cells producing intracellular Arginase 1 are myeloid cells. In one embodiment, the cells producing intracellular Arginase 1 are CD14-, CD16-.

[0168] In one embodiment, the cells producing intracellular Arginase 1 constitutively express Arginase 1.

[0169] The term "determining" refers to "detecting" or "measuring" the level or presence or absence of an item (e.g., a cell type) to be detected. As used herein, the term "level" refers to the amount, number, percentage, or concentration of an item.

[0170] As mentioned above, the inventors have observed that T cells that respond to immune checkpoint inhibitors are outside the tumor microenvironment.Therefore, the sample is a sample from tissue outside the tumor microenvironment.In this context, 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 or polynuclear cells.

[0171] If a subject is assessed or identified as non-responsive to immune checkpoint inhibitor therapy, the non-responsiveness can be at least partially reversed by administering to the subject an effective amount of an agent that inhibits Arginase 1 activity and / or an agent that targets cells that express Arginase 1.

[0172] Another aspect provides a method of increasing responsiveness to immune checkpoint inhibitor treatment in a subject suffering from a cancer non-responsive to immune checkpoint inhibitor treatment, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor.

[0173] Another embodiment is a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, each response control representing a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by each cell of the sample, cells having the same expression of the group of biomarkers being classified into the same class, and the group of biomarkers being 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, as well as 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) providing a marker comprising at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers selected from: TIGIT, CD38, Ki67, Granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells of a sample from a subject within a series of classes of positive and negative reaction controls, wherein the sample is in the form of a sample of cells from the subject's blood; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to have a likelihood of being non-responsive to the treatment if the distribution of cells of the sample within the set of classes from the subject is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing the likelihood that the subject will be non-responsive to treatment with an immune checkpoint inhibitor, The method provides a method for determining whether a subject is non-responsive to an immune checkpoint inhibitor by:

[0174] In one embodiment, the group of biomarkers comprises at least 13 markers selected from 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 beta7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0175] Another embodiment is a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and / or negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of blood samples from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by each cell of the sample, cells having the same expression of the group of biomarkers being classified into the same class, and the group of biomarkers being 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, as well as 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) providing a marker comprising at least five markers, typically at least six, seven, eight, nine, ten, eleven, twelve or at least thirteen markers selected from: TIGIT, CD38, Ki67, Granzyme B, CD134 (OX40), CD192 (CCR2), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), integrin beta 7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, GM-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells of a sample from the subject within a series of classes of positive and / or negative control responses, wherein the sample is in the form of a sample of cells from the subject's blood; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to have a likelihood of being non-responsive to the treatment if the distribution of cells of the sample within the set of classes from the subject is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing the likelihood that the subject will be non-responsive to treatment with an immune checkpoint inhibitor, The method provides a method for determining whether a subject is non-responsive to an immune checkpoint inhibitor by:

[0176] In one embodiment, the group of biomarkers comprises at least 13 markers selected from 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 beta7, CLA, CD183 (CXCR3), CD185, (CXCR5), CCR10, and intracellular arginase 1.

[0177] In one embodiment, (ii) determining the distribution of cells of the sample within a set of classes comprises: a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes to determine the distribution of cells of a sample from a subject within a series of classes of response controls.

[0178] In one embodiment, (iii) comparing the distribution of cells of the sample from the subject within a set of classes comprises: comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; to determine whether the distribution of cells of the sample within a set of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0179] As described above, the present inventors have demonstrated that cells in the blood of subjects associated with non-responsiveness to immune checkpoint therapy express ARG1 + The present inventors further characterized these cells and found that they are a unique cell population that exhibits a phenotype distinct 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-, and CD66b-.

[0180] One embodiment provides a method for determining whether a subject is non-responsive 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 the subject is determined to be non-responsive to treatment if the level of cells that are ARG1+, CD14-, CD16-, CD66b- in the sample is elevated compared to a positive response control.

[0181] A further aspect is a method of treating cancer in a human subject, comprising: (b) assessing whether the subject is non-responsive 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; (c) assessing whether the level of ARG1+, CD14-, CD16-, CD66b- cells in the sample is elevated compared to a positive control; and determining that the subject is non-responsive to the treatment. (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; The present invention provides a method comprising:

[0182] A further embodiment is a method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: determining the level of ARG1+, CD14-, CD16-, CD66b- cells in a blood sample from the subject, wherein the subject is determined to be non-responsive to treatment if the level of ARG1+, CD14-, CD16-, CD66b- cells in the sample is elevated compared to a positive response control. The method provides a method for determining whether a subject is non-responsive to an immune checkpoint inhibitor by:

[0183] In one embodiment, the positive response control is the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from a subject or subjects who are responsive to treatment with an immune checkpoint inhibitor.

[0184] In various embodiments, cells that are ARG1+, CD14-, CD16-, CD66b have the following phenotype: (a) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, (b) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, (c) ARG1+, CD14-, CD16-, CD66b-, HLA-DR-, CD123+, CD45RO+, CD38+, (d) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, (e) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, (f) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, (g) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, (h) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33lo, (i) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33lo, CD192(CCR2)int, (j) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33lo, CD45lo, (k) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33lo, CD45lo, CD56-, (l) ARG1+, CD3-, CD14-, CD16-, CD66b-, CD19-, HLA-DR-, CD123+, CD45RO+, CD38+, CD304-, GM-CSFRα chain-, CD15-, CD33lo, CD45lo, CD56-, CD20- It has.

[0185] A further embodiment provides a method of treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, and thereafter administering an effective amount of the immune checkpoint inhibitor, optionally in combination with further administration of the agent that inhibits arginase 1 activity and / or the agent that targets cells that express arginase 1.

[0186] As used herein, the term "effective amount" refers to the dose of a drug or other active substance that produces the therapeutic effect of its administration.The exact dose depends on the purpose of treatment and can 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, the "effective amount" of an active substance that inhibits arginase 1 activity and / or targets cells that express arginase 1 is the amount of active substance that is effective in making a subject responsive to immune checkpoint inhibitors.

[0187] The agent that inhibits Arginase 1 activity and / or targets cells that express Arginase 1 is administered for a time sufficient to render the subject responsive to an immune checkpoint inhibitor.

[0188] A subject can be assessed as being responsive to an immune checkpoint inhibitor, for example, using the methods described in WO2019 / 051542.

[0189] In one embodiment, a subject, after treatment with an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1, determining a distribution of cells in a sample from a subject administered an agent within a series of classes of positive and / or negative control responses, the sample being in the form of a sample of cells from the subject's blood; comparing the distribution of cells of the sample from the subject within the series of classes with a negative response control and / or a positive response control, wherein the subject is determined to have a likelihood of being responsive to the treatment if the distribution of cells of the sample within the series of classes from the subject is substantially the same as the distribution of cells within the series of classes represented by the positive response control and / or is not the same as the distribution of cells within the series of classes represented by the negative response control; Responsiveness to an immune checkpoint inhibitor can be assessed by a method comprising:

[0190] Thus, the length of time required for administration of an agent that inhibits Arginase 1 activity and / or targets cells that express Arginase 1 can be monitored by assessment at intervals following administration of the agent.

[0191] 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-boronohexanoic 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-iodoacetamidopentanoic acid. , NG-hydroxy-L-arginine, (2S)-(+)-amino-6-iodoacetamidohexanoic acid, and (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, CB-1158 (numidardistat), OATD-02 (WO 2017191130), or compounds described in WO 2019173188, WO 2019177873, and WO 2020161675.

[0192] In one embodiment, the agent inhibits intracellular arginase 1 and / or targets cells that express high levels of intracellular arginase 1.

[0193] In one embodiment, the agent that targets cells that express Arginase 1 targets cells that constitutively express Arginase 1.

[0194] In embodiments in which the agent targets cells that express arginase, the agent may be an antibody that specifically binds to and typically inactivates cells that express Arginase 1.

[0195] In another embodiment, the agent that inhibits Arginase 1 activity and / or targets cells that express Arginase 1 is an agent that reduces or inhibits Arginase gene expression by inhibiting either transcription or translation. For example, an agent that inhibits arginase activity can be, for example, a gene silencing nucleic acid that targets arginase mRNA, such as an siRNA agent (see, e.g., WO / 44895, WO 99 / 32619, WO 01 / 75164, WO 01 / 92513, WO 01 / 29058, WO 01 / 89304, WO 02 / 16620, and WO 02 / 29858), a CRISPR agent (see, e.g., 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.

[0196] Typically, the dosage of an 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, ranges from 1 μg to about 100 g per kilogram of the subject's body weight. The exact dosage administered will vary depending on various factors, including, for example, the type of cancer condition 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, body weight, general health, sex, diet, method and time of administration, excretion rate, drug combination, the severity of the particular condition, and the subject receiving therapy.

[0197] The agent may be administered as frequently as several times daily, or less frequently, such as once daily, once weekly, once every two weeks, once monthly, or even less frequently, such as once every few months, or even once a year or less. The frequency of administration will be readily apparent to one of skill in the art and will depend on a variety of factors, including, but not limited to, the type and severity of the disease being treated, the species and age of the animal, etc.

[0198] As used herein, an "immune checkpoint inhibitor" refers to a molecule or agent that inhibits immune checkpoint-mediated immunosuppression. The checkpoint inhibitor can be an inhibitor of any checkpoint that inhibits T cell function and is used in cancer therapy. 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 cemiplimab. Examples of anti-PD-L1 antibodies include atezolizumab, avelumab, and durvalumab.

[0199] In another embodiment, the checkpoint inhibitor is an inhibitor of the binding of CTLA-4 to its ligand. In one embodiment, the checkpoint inhibitor that is an inhibitor of the interaction of CTLA-4 with its ligand is an anti-CTLA-4 antibody. One example of an anti-CTLA-4 antibody used in cancer therapy is ipilimumab.

[0200] 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), LAG 3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin domain and mucin domain 3) and VISTA (V domain Ig suppressor of T cell activation).

[0201] Cancer can be any cancer that can be treated with immune checkpoint inhibitor in the subject who responds to treatment.The example of cancer that can be treated using the method described herein includes bladder cancer, colon cancer, breast cancer, prostate cancer, renal 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).

[0202] Another aspect provides a pharmaceutical composition comprising an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier.

[0203] By "pharmaceutically acceptable carrier" is meant one that is compatible with the other ingredients of the composition and not harmful to the subject. The compositions may contain other therapeutic agents, as described below, and can be formulated according to techniques well known in the art of pharmaceutical formulation using, for example, conventional liquid vehicles or diluents and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) of a type appropriate to the desired method of administration (see, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

[0204] Pharmaceutical compositions are usually in the form of sterile injectable aqueous suspensions. These suspensions may be formulated according to known techniques and contain the active ingredient mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and arabic gum, and dispersing or wetting agents may be naturally occurring phospholipids such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxyethanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, 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, such as ethyl or n-propyl p-hydroxybenzoate.

[0205] Sterile injectable preparations can be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectable preparations.

[0206] A further aspect is a kit for treating cancer in a subject, comprising: (a) an agent that inhibits arginase activity in ARG1+ cells or an agent that targets cells that express arginase; and optionally, (b) Immune checkpoint inhibitors and A kit is provided, comprising:

[0207] The kit may further include instructions for use.

[0208] Generally, the term "treating" means acting on a subject, tissue, or cell to achieve a desired pharmacological and / or physiological effect, including (a) preventing the onset of disease in a subject who may be predisposed to, but has not yet been diagnosed with, the disease, (b) inhibiting the disease, i.e., halting its progression, or (c) alleviating or ameliorating the effects of the disease, i.e., causing a reduction in the effects of the disease. In one embodiment, treatment achieves the result of reducing the number of cancer cells in the recipient subject.

[0209] The term "subject" refers to any animal having a disease that requires treatment by the present method. In the context of this disclosure, the subject is a human.

[0210] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited solely by the appended claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0211] 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.

[0212] All documents mentioned in this specification are incorporated herein by reference. It will be understood by those skilled in the art that many variations and / or modifications can be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments, therefore, should be considered in all respects as illustrative and not restrictive.

[0213] In the following claims and in the foregoing description of the invention, unless the context requires otherwise by express words or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., to specify the presence of stated features but not to exclude the presence or addition of further features in various embodiments of the invention.

[0214] The following non-limiting examples are provided to illustrate and so that the nature of the present invention may be more clearly understood.

[0215] Example Peripheral blood samples are obtained by venipuncture and collected in anticoagulant tubes (e.g., those containing EDTA or heparin). Leukocytes are purified using Ficoll-Hypaque density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs), which are then either immediately prepared for cytometry or frozen for long-term storage, e.g., at -80°C or in liquid nitrogen.

[0216] Cryopreserved leukocytes are thawed using conventional techniques prior to analysis.

[0217] Cellular distribution is determined using techniques that identify the expression of a given protein or transcript in each single cell within a sample. Typically, cells are treated with a mixture of 10–40 antibodies, each specific for an individual cellular protein, and labeled with a unique metal isotope or fluorescent dye so that the treated sample can be analyzed using mass cytometry or fluorescence cytometry, respectively.

[0218] Pretreatment PBMCs were analyzed by mass cytometry in a cohort of 10 stage 4 non-small cell lung cancer (NSCLC) patients scheduled for anti-PD-1 / PD-L1 therapy. To determine immune signatures, we analyzed data from 13 mAbs (specific for CD45, CD3, CD4, CD8, CD45RO, CD197 (CCR7), CD25, CD127, CD196 (CCR6), integrin β7, CD56, CD16, and CD14) from a panel of 41 mAbs. Five patients demonstrated clinical responses to anti-PD-1 / PD-L1 therapy, and five patients experienced disease progression despite anti-PD-1 / PD-L1 therapy. The pretreatment blood immune signatures for anti-PD-1 / PD-L1 therapy are shown in Figure 1. Hierarchical clustering confirmed the usefulness of the pretreatment blood signature in distinguishing responders from non-responders.

[0219] Table 2 defines the cell populations shown in Figure 1. 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.

[0220] [Table 3]

[0221] Intracellular detection of arginase 1 using a metal-conjugated mAb specific for human arginase 1 revealed a distinct population of arginase 1-expressing cells within the total PBMC population, as shown in Figure 2. Cells constitutively expressing high levels of arginase 1 in pretreatment PBMCs from NSCLC patients were negative for HLA-DR, CD14, CD16, and CD66b, but positive for CD45RO and CD38. Monocytic MDSCs (M-MDSCs, HLA-DRlo CD14+) and neutrophils (PMN-MDSCs, HLA-DRlo CD16+CD66b+) have been reported to express arginase 1, but no intracellular arginase 1 expression was detected in M-MDSCs or PMN-MDSCs in the unstimulated pretreatment 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 reported in previous studies.

[0222] In 24 NSCLC patients, a statistically significant negative correlation was observed between the number of cells expressing intracellular arginase 1 in pretreatment PBMCs and clinical response to anti-PD-1 / PD-L1 therapy (Figure 3).

[0223] In a cohort of 21 NSCLC patients for whom pretreatment plasma samples were available, we measured plasma arginase activity (using a colorimetric assay measuring the conversion of L-arginine to L-ornithine) and plasma L-arginine (using mass spectrometry). As expected, plasma arginine activity was higher in samples with lower arginine content (Figure 5), but there was no statistically significant difference between responder and non-responder patients (Figure 4). These results strongly support the model that intracellularly expressed arginase is a major driver of reduced T cell responses to anti-PD-1 / PD-L1 therapy, preventing effective clinical responses.

[0224] These data indicate that therapies targeting the cells that make arginase, rather than circulating arginase, are effective in converting non-responder patients to a responder state.

[0225] Markers associated with arginase 1-expressing cells A panel of monoclonal antibodies was used to obtain a complete definition of the phenotype of Arginase 1-expressing cells and how these cells differ from other circulating myeloid cells. The results are shown in Table 3.

[0226] [Table 4]

[0227] Arginase 1 expression in myeloid-derived suppressor cells (MDSCs) has been reported in both neutrophilic PMN-MDSCs and monocytic mo-MDSCs. The arginase 1-expressing cells described herein differ from both types of MDSCs. They are negative for CD14, which is used to identify mo-MDSCs, and negative for CD15, CD16, and CD66b, which are used to identify PMN-MDSCs. They express CD123, which is not expressed on MDSCs. CD123 is also highly expressed on plasmacytoid DCs (pDCs). The arginase 1-expressing cells described herein differ from pDCs because they are negative for HLA-DR, which pDCs express. CD123 is also highly expressed on basophils. The arginase 1-expressing cells described herein differ from basophils because basophils do not express arginase 1.

[0228] In further experiments to assess the morphology of arginase 1-expressing cells, arginase 1-expressing PBMCs from NSCLC patients were sorted using a combination of mAbs against CD45, HLA-DR, CD11b, CD123, arginase 1, CD14, CD15, and lineage (CD19, CD3, and CD56) antibodies. For comparison, pDCs, neutrophils, and monocytes were also sorted. Sorted cells were cytospun onto glass slides and stained with Giemsa. Analysis of nuclear morphology revealed that arginase 1-positive cells had a distinctive morphology, usually with bilobed or trilobed segmented nuclei. Nuclei of many arginase 1-expressing cells exhibited a cerebriform appearance, while a minority of cells had a cleaved appearance. The cells did not contain basophilic granules typical of basophils. A second population of CD123+HLA-DR-Arginase 1-negative cells (0.23% of CD45+ cells) was smaller in size and had a lymphoid appearance, likely representing the ILC-like population described in association with psoriasis in Mora-Velandia et al., 2017, Front. Immunol. 8:176.

[0229] In addition to NSCLC, where arginase 1-expressing cells in the blood were found more frequently than in healthy controls and correlated with early resistance to treatment with immunotherapy targeting the PD-1 / PD-L1 pathway, we also demonstrated that these cells were abnormally abundant in patients with colorectal cancer, where arginase 1-expressing cells were absent in cell mixtures of dissociated cancer samples from the same patients.

[0230] Targeting arginase 1-positive cells to restore and reverse non-responsiveness to anti-PD-1 / PD-L1 therapy We propose that the use of agents targeting arginase 1-positive cells will revert the non-responsive immune signature to normal over time. However, if arginase 1-targeted therapy can restore potential cancer-regulatory T cells to normal signaling function before anti-PD-1 / PD-L1 therapy is initiated, it may not be necessary to wait long enough to revert the signature, even before other aspects of the immune signature, such as the lack of naive T cells, normalize.

[0231] This specification also describes the following items 1 to 20: 1. A method of treating cancer in a human subject, comprising: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by the cells of the sample, and cells having the same expression of biomarkers from the group of biomarkers being classified into the same class, wherein the group of biomarkers is CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, providing a marker comprising at least 13 markers selected from 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; (ii) determining the distribution of cells in a blood sample from the subject within a series of classes of positive and negative reaction controls; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control; assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor; (b) if the subject is determined to be non-responsive to treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of an immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1; A method comprising:

[0232] 2. The method of item 1, wherein the group of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0233] 3. (ii) Determining the distribution of cells in a sample within a set of classes is a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes 3. The method of claim 1 or 2, comprising determining the distribution of cells of a sample from a subject within a series of classes of response controls.

[0234] 4. (iii) Comparing the distribution of cells in samples from subjects within a set of classes; comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; 2. The method of claim 1, comprising determining whether the distribution of cells of the sample within a series of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0235] 5. The method of any one of items 1 to 4, wherein the sample is a peripheral blood sample.

[0236] 6. The method of any one of items 1 to 5, 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 1 before the treatment.

[0237] 7. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-nonresponsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: (a) (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, wherein each response control represents a distribution of cells of a blood sample from the positive responders and non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by each cell of the sample, wherein cells having the same expression of the group of biomarkers are classified into the same class, and the group 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, as well as CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD11b, CD11c, CD66b, CD304, FoxP3, CD127, CD25, CD56, HLA-DR, IgD, CD27, CD86, FCRL3, providing a marker comprising at least 13 markers selected from 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; (ii) determining the distribution of cells of a sample from a subject within a series of classes of positive and negative reaction controls, wherein the sample is in the form of a sample of cells from the subject's blood; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with a negative response control and / or a positive response control, wherein the subject is determined to have a likelihood of being non-responsive to the treatment if the distribution of cells of the sample within the set of classes from the subject is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing the likelihood that the subject will be non-responsive to treatment with an immune checkpoint inhibitor, The method of claim 1, wherein the patient is determined to be non-responsive to an immune checkpoint inhibitor by

[0238] 8. The method of item 7, wherein the group of biomarkers comprises 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 13 markers selected from CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

[0239] 9. (ii) Determining the distribution of cells in a sample within a set of classes is a. assessing each cell of a sample from a subject for expression of each biomarker of a panel of response control biomarkers; b. classifying each cell of a sample from the subject into one of a set of classes of response controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes 9. The method of claim 7 or 8, comprising determining the distribution of cells of a sample from a subject within a series of classes of response controls.

[0240] 10. (iii) Comparing the distribution of cells in samples from subjects within a set of classes is comparing the measured cell counts of the sample from the subject in each class of the series of classes of response controls with the cell counts of the response controls in each class of the series of classes of response controls; 8. The method of item 7, comprising determining whether the distribution of cells of the sample within a series of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

[0241] 11. The method of any one of items 7 to 10, wherein the sample is a peripheral blood sample.

[0242] 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 treatment compared to the level of cells expressing intracellular arginase before treatment.

[0243] 13. A method of treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase activity or an agent that targets cells that express arginase for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, and administering the effective amount of the immune checkpoint inhibitor, optionally in combination with a further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1.

[0244] 14. The method of any one of items 1 to 13, wherein the immune checkpoint inhibitor is an inhibitor of the interaction of PD-1 with its ligand.

[0245] 15. The method of any one of items 1 to 14, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0246] 16. The method of 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.

[0247] 17. The method of any one of items 1 to 16, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, durvalumab, atezolizumab, or avelumab.

[0248] 18. The method of any one of paragraphs 1 to 17, wherein the agent that targets cells that express Arginase 1 targets cells that constitutively express Arginase 1.

[0249] 19. A pharmaceutical composition comprising an agent that inhibits arginase 1 activity or targets cells that express arginase 1, and an immune checkpoint inhibitor.

[0250] 20. A kit for treating cancer, comprising: (a) an agent that inhibits arginase 1 activity or targets cells that express arginase 1; and, optionally, (b) Immune checkpoint inhibitors and Includes a kit.

[0251] This application claims priority from Australian Provisional Patent Application No. 2022903578, which is incorporated herein by reference in its entirety.

Claims

1. 1. A method of treating cancer in a subject identified as non-responsive to treatment with an immune checkpoint inhibitor, comprising: (a) administering an effective amount of an agent that inhibits Arginase 1 activity and / or an agent that targets cells that express Arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (b) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1; wherein the subject (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, wherein each response control represents a distribution of cells of blood samples from the positive responders and the non-responders within a set of classes, the distribution of cells according to the expression of a group of biomarkers by cells of the sample, wherein cells having the same expression of biomarkers from the group of biomarkers are classified into the same class, and the group of biomarkers is selected from the group consisting of CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD17, CD18, CD29, CD30, CD41, CD42, CD43, CD44, CD8, CD19, CD20, CD14, CD15, CD16, CD19, CD29, CD29, CD30, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD providing a marker comprising at least five markers selected from D11b, 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-CSFR alpha chain, and intracellular arginase 1; (ii) determining the distribution of cells in the blood sample from the subject within a series of classes of the positive reaction control and the negative reaction control; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with the negative response control and / or the positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. The method of claim 1, wherein the patient is identified as non-responsive to said immune checkpoint inhibitor by

2. 1. A method of treating cancer in a human subject, comprising: (a) (i) providing positive response controls from one or more positive responders who have experienced a clinical response to the treatment and negative response controls from one or more non-responders who have not experienced a clinical response to the treatment, wherein each response control represents a distribution of cells of a blood sample from said positive responder and said non-responder within a set of classes, said distribution of cells according to the expression of a group of biomarkers by cells of said sample, wherein cells having the same expression of biomarkers from said group of biomarkers are classified into the same class, said group of biomarkers being CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD19, CD20, CD14, CD15, CD16, CD17, CD19, CD20, CD19, CD20, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45 ... 1b, 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-CSFR alpha chain and intracellular arginase 1; (ii) determining the distribution of cells in the blood sample from the subject within a series of classes of the positive reaction control and the negative reaction control; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with the negative response control and / or the positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor; (b) if the subject is determined to be non-responsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1; A method comprising:

3. The group of biomarkers is 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 3. The method of claim 1 or 2, wherein the markers comprise at least 13 markers selected from the group consisting of 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. 4. The method of any one of claims 1 to 3, wherein the group of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

5. 4. The method of any one of claims 1 to 3, wherein the group of biomarkers comprises at least 13 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 β7, GM-CSFR α chain, and intracellular arginase 1.

6. 4. The method of any one of claims 1 to 3, wherein the group of biomarkers comprises at least 13 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, and intracellular arginase 1.

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

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

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

11. (ii) determining the distribution of cells of the sample within said set of classes; d. assessing the expression of each biomarker in the group of biomarkers in each cell of the sample from the subject; e. classifying each cell of the sample from said subject into one of said set of classes of reaction controls; f. Measuring the number of cells in the test sample in each class of the series of reaction control classes.

11. The method of claim 1, wherein the distribution of cells of a sample from the subject within a series of classes of the reaction control is determined.

12. (iii) comparing the distribution of cells of the sample from the subject within the set of classes; comparing the measured cell counts of the sample from the subject in each class of the series of classes of reaction controls with the cell counts of the reaction controls in each class of the series of classes of reaction controls.

12. The method of claim 1, further comprising determining whether the distribution of cells of the sample within a series of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

13. 13. The method of any one of claims 1 to 6 or claim 11 or 12, wherein the set of classes comprises naive Tcon in CD4+, CD4+ Treg in live cells, CD4+ Tcon in live cells, β7+ in CD45RO+ CD4+ Tcon, CCR6+ in CD45RO+ CD4+ Tcon, naive CD8+ in live cells, naive CD8+ in live cells, CD45RO+ in CD8+, CD16loCD56hi NK in live cells, CD14+CD16- classical monocytes in live cells, CD4+ in live cells, CD8+ in live cells, naive in CD8+.

14. 13. The method of any one of claims 1 to 6 or claim 11 or 12, wherein the set of classes comprises the classes of % CD8+ naive to total, % CD8+ to total, % CD8+CD45RO- naive to total, % B cells to total, % CD3+ to total, % CD4+ to total, % CD4 naive to total, % CD4 naive to CD4, % CD4+ TCM to total, % Treg to total, % Treg CD45RO+ to total, % IntB7 to RO, % CD8+CD45RO+ memory to CD8, % NK cells to CD3-CD20-, % CCR6 to RO, % monocytes to total, % CD14 monocytes to total, and % NK cells to total.

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

16. 16. The method of any one of claims 1 to 15, further comprising monitoring the effectiveness of the treatment, comprising 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. 1. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: (a) (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, wherein each response control represents a distribution of cells of a blood sample from the positive responder and the non-responder within a set of classes, the distribution of cells according to the expression of a group of biomarkers by each cell of the sample, wherein cells having the same expression of the group of biomarkers are classified into the same class, the group of biomarkers being 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-CSFR alpha chain, and intracellular arginase 1; (ii) determining the distribution of cells of a sample from the subject within a series of classes of the positive reaction control and the negative reaction control, wherein the sample is in the form of a sample of cells from the blood of the subject; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with the negative response control and / or the positive response control, wherein the subject is determined to have a likelihood of being non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. assessing the likelihood that the subject will be non-responsive to treatment with an immune checkpoint inhibitor, and determining that the subject is non-responsive to the immune checkpoint inhibitor by

18. The group of biomarkers is 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), TIGI 18. The method of claim 17, wherein the markers comprise at least 13 markers selected from T, 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. 18. The method of claim 17, wherein the group of biomarkers comprises the markers CD45, CD45RO, CD3, CD4, CD8, CD14, CD16, CD127, CD25, CD56, CD196 (CCR6), CD197 (CCR7) and integrin β7.

20. 18. The method of claim 17, wherein the group of biomarkers comprises at least 13 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 β7, GM-CSFR α chain, and intracellular arginase 1.

21. 18. The method of claim 17, wherein the group of biomarkers comprises at least 13 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, and intracellular arginase 1.

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

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

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

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

26. (ii) determining the distribution of cells of the sample within said set of classes; d. assessing the expression of each biomarker in the group of biomarkers in each cell of the sample from the subject; e. classifying each cell of the sample from said subject into one of said set of classes of reaction controls; f. Measuring the number of cells in the test sample in each class of the series of reaction control classes.

26. The method of claim 17, wherein the method comprises determining a distribution of cells of a sample from the subject within a series of classes of the reaction control.

27. (iii) comparing the distribution of cells of the sample from the subject within the set of classes; comparing the measured cell counts of the sample from the subject in each class of the series of classes of reaction controls with the cell counts of the reaction controls in each class of the series of classes of reaction controls.

26. The method of claim 17, further comprising determining whether the distribution of cells of the sample within a series of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

28. 28. The method of any one of claims 17 to 27, wherein the set of classes comprises naive Tcon in CD4+, CD4+ Treg in live cells, CD4+ Tcon in live cells, β7+ in CD45RO+ CD4+ Tcon, CCR6+ in CD45RO+ CD4+ Tcon, naive CD8+ in live cells, naive CD8+ in live cells, CD45RO+ in CD8+, CD16loCD56hi NK in live cells, CD14+CD16- classical monocytes in live cells, CD4+ in live cells, CD8+ in live cells, and naive in CD8+.

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

30. 30. The method of any one of claims 17 to 29, 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.

31. 1. A method of treating cancer in a subject non-responsive to treatment with an immune checkpoint inhibitor, comprising administering to the subject an effective amount of an agent that inhibits arginase activity or an agent that targets cells that express arginase, for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, and administering the effective amount of the immune checkpoint inhibitor, optionally in combination with a further administration of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1.

32. 1. A method of treating cancer in a human subject, comprising: (d) assessing whether the subject is non-responsive to treatment with an immune checkpoint inhibitor, comprising determining the level of cells that express intracellular arginase 1 (ARG1+) and are CD14-, CD16-, and CD66b- in a blood sample from the subject; (e) assessing whether the subject is determined to be non-responsive to treatment if the level of cells expressing intracellular arginase 1 (ARG1+) in the sample is elevated compared to a positive control; (b) if the subject is determined to be non-responsive to the treatment, administering an effective amount of an agent that inhibits arginase 1 activity and / or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor; (c) administering an effective amount of said immune checkpoint inhibitor, optionally in combination with further administration of an agent that inhibits arginase 1 activity and / or targets cells that express arginase 1; A method comprising:

33. 1. A method of increasing responsiveness to treatment with an immune checkpoint inhibitor in a subject suffering from a treatment-non-responsive cancer, comprising administering to the subject an effective amount of an agent that inhibits arginase 1 activity or an agent that targets cells that express arginase 1 for a time sufficient to render the subject responsive to the immune checkpoint inhibitor, wherein the subject: 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 which are CD14-, CD16-, CD66b- in the sample is elevated compared to a positive control. and determining that the subject is non-responsive to the immune checkpoint inhibitor by

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

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

44. 44. The method of any one of claims 1 to 43, wherein the agent that targets cells that express Arginase 1 targets cells that constitutively express Arginase 1.

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

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

47. 1. A method for determining whether a subject suffering from cancer is non-responsive to treatment with an immune checkpoint inhibitor, 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, wherein each response control represents a distribution of cells of a blood sample from the positive responder and the non-responder within a set of classes, the distribution of cells according to the expression of a group of biomarkers by cells of the sample, wherein cells having the same expression of biomarkers from the group of biomarkers are classified into the same class, and the group of biomarkers includes intracellular arginase 1, as well as 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, and GM-CSFR alpha chain; (ii) determining the distribution of cells in the blood sample from the subject within a series of classes of the positive reaction control and the negative reaction control; (iii) comparing the distribution of cells of the sample from the subject within the set of classes with the negative response control and / or the positive response control, wherein the subject is determined to be non-responsive to the treatment if the distribution of cells of the sample from the subject within the set of classes is substantially the same as the distribution of cells within the set of classes represented by the negative response control and / or is not the same as the distribution of cells within the set of classes represented by the positive response control. A method comprising:

48. 48. The method of claim 47, wherein the group of biomarkers comprises intracellular arginase 1 and at least eight markers selected from 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. 48. The method of claim 47, wherein the group of biomarkers comprises intracellular arginase 1 and at least thirteen markers selected from CD45, CD3, CD20, CD14, CD15, CD16, CD11b, CD19, CD66b, CD304, CD123, CD33, CD56, HLA-DR, CD38, CD192 (CCR2), and GM-CSFR alpha chain.

50. 48. The method of claim 47, wherein the group of biomarkers comprises intracellular arginase 1, as well as CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19, CD3, and CD56.

51. 48. The method of claim 47, wherein the group of biomarkers comprises intracellular arginase 1, as well as CD3, HLA-DR, CD14, CD16, CD19, CD66b, CD45RO, and CD38.

52. 48. The method of claim 47, wherein the group of biomarkers comprises intracellular arginase 1, as well as CD3, CD45, HLA-DR, CD11b, CD123, CD14, CD15, CD19 and CD56.

53. (ii) determining the distribution of cells of the sample within said set of classes; assessing each cell of a sample from said subject for expression of each biomarker of said panel of biomarkers in said response controls; b. classifying each cell of the sample from said subject into one of said set of classes of reaction controls; c. Measuring the number of cells in the test sample in each class of the series of reaction control classes.

53. The method of any one of claims 47 to 52, comprising determining a distribution of cells of a sample from said subject within a series of classes of said reaction controls.

54. (iii) comparing the distribution of cells of the sample from the subject within the set of classes; comparing the measured cell counts of the sample from the subject in each class of the series of classes of reaction controls with the cell counts of the reaction controls in each class of the series of classes of reaction controls.

54. The method of claim 53, comprising determining whether the distribution of cells of the sample within the set of classes of reaction controls is the same as the distribution of cells represented by one of the reaction controls.

55. 55. The method of any one of claims 47 to 54, wherein the set of classes comprises the classes of % CD8+ naive to total, % CD8+ to total, % CD8+CD45RO- naive to total, % B cells to total, % CD3+ to total, % CD4+ to total, % CD4 naive to total, % CD4 naive to CD4, % CD4+ TCM to total, % Treg to total, % Treg CD45RO+ to total, % IntB7 to RO, % CD8+CD45RO+ memory to CD8, % NK cells to CD3-CD20-, % CCR6 to RO, % monocytes to total, % CD14 monocytes to total, and % NK cells to total.

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

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

58. 1. A method for determining whether a subject is non-responsive to treatment with an immune checkpoint inhibitor, comprising determining the level of ARG1+, CD14-, CD16-, CD66b- cells in a blood sample from the subject, wherein the subject is determined to be non-responsive to the treatment if the level of ARG1+, CD14-, CD16-, CD66b- cells in the sample is elevated compared to a positive reaction control.

59. 59. The method of claim 58, wherein said positive response control is the level of cells that are ARG1+, CD14-, CD16-, CD66b- in a blood sample from a subject or subjects responsive to treatment with said immune checkpoint inhibitor.

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+ cells.

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

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

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