Methods and agents for assessing T cell function and predicting response to therapy

Abstract

The present disclosure generally relates to methods and agents / reagents for assessing T cell function and for predicting responses to therapy. More specifically, the present invention relates to methods and agents / reagents for detecting different forms of Eomesodermal Protein (EOMES) in T cells, which can be used to assess T cell function, assess the immune function of a subject, predict the likelihood of a cancer patient's response to therapy (including immunotherapy), stratify cancer patients into likely responders or non-responders to therapy, and manage the treatment of cancer patients.

Description

Technical Field

[0001] The present disclosure generally relates to methods and agents / reagents for assessing T cell function and predicting responses to therapy. More specifically, the present invention relates to methods and agents / reagents for detecting different forms of Eomesodermin (EOMES) in T cells, which can be used to assess T cell function, assess the immune function of a subject, predict the likelihood of a cancer patient's response to therapy (including immunotherapy), stratify cancer patients into possible responders or non-responders to therapy, and manage the treatment of cancer patients.

[0002] Related applications

[0003] This application claims priority to Australian provisional application number 2019900628 filed on February 27, 2019, entitled “Methods and agents for assessing T cell function and predicting response to therapy”, the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] Cancer is a significant cause of morbidity and mortality worldwide. Although the standard of care for many different cancer types has improved significantly over the years, the current standard of care continues to fall short of meeting the need for effective therapies to improve cancer treatment. The clinical application of immuno-oncology agents targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death receptor-1 (PD-1) and its ligand PD-L1 has led to improvements in the standard of care for many cancer types. While these checkpoint inhibitors produce improved clinical responses in some cancers, durable clinical responses occur in only approximately 10–45% of patients. Furthermore, a significant number of tumors either develop resistance or become refractory. For example, approximately 20–50% of melanomas and lung cancers respond significantly to immunotherapy, while others do not. Therefore, determining which subjects are more appropriate candidates for cancer therapy is highly advantageous from the perspective of both medical care and patient quality of life.

[0005] Accordingly, there remains a need in the art for methods and agents / reagents that help predict response to therapy, including immunotherapy, with improved accuracy. Summary of the Invention

[0006] The present invention is partly derived from determining that different post-translational modifications of EOMES in T cells are related to the localization of the transcription factor to different cellular compartments. Moreover, different post-translational modifications are related to different T cell functions and different responsiveness to cancer therapy. In particular, the inventors have found that different post-translational modifications of lysine (i.e., EOMES-641K) in the nuclear localization sequence (NLS) of EOMES are related to whether EOMES is located in the nucleus or the cytoplasm. More specifically, it was found that the acetylation of EOMES-641K (i.e., EOMES-263K-Ac) is related to the biased localization of EOMES to the nucleus. Interestingly, it was found that this acetylated form of EOMES is also related to T cell exhaustion and resistance or non-responsiveness to therapy. In contrast, it was found that the methylation of EOMES-641K (i.e., EOMES-641K-Me) is generally related to the more biased localization of EOMES to the cytoplasm of T cells. This form of EOMES is also associated with competent immune T cells and responsiveness to cancer therapy. The inventors have also demonstrated that post-translational modification of lysine (i.e., EOMES-373K) in the DNA binding domain of EOMES is associated with responsiveness to cancer therapy. Specifically, methylation of EOMES-373K (i.e., EOMES-373K-Me) is associated with cytoplasmic localization and responsiveness to cancer therapy.

[0007] As described below, these findings have been reduced to methodological practices and reagents / reagents for assessing T cell function, for assessing immune function in a subject, for predicting and / or monitoring response to therapy, for stratifying patients into likely responders or non-responders based on the expression pattern of EOMES in T cells, for managing patient treatment based on stratification, and for predicting clinical outcomes.

[0008] Thus, in one aspect, provided herein is a method for assessing T cell function comprising, consisting of, or consisting essentially of detecting post-translational modifications in the nuclear localization sequence and / or DNA binding motif of EOMES in T cells.

[0009] In one embodiment, the method includes detecting acetylation of EOMES-641K (also referred to herein as "EOMES-641K-Ac") in T cells and determining that the T cells are dysfunctional. In a specific embodiment, an elevated level of EOMES-641K-Ac relative to a suitable control (e.g., functional T cells) is detected in the T cells. The method may also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-641K-Ac in the T cells. In a specific example, the method includes detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Ac in the T cells or the ratio of cytoplasmic to nuclear localization.

[0010] In another embodiment of the method for assessing T cell function, the method includes detecting methylation of EOMES-641K (also referred to herein as "EOMES-641K-Me") in T cells and determining that the T cells are functional. For example, an elevated level of EOMES-641K-Me relative to a suitable control (e.g., dysfunctional T cells) can be detected in T cells. The method can also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-641K-Me in T cells. In one example, the ratio of nuclear to cytoplasmic localization of EOMES-641K-Me in T cells or the ratio of cytoplasmic to nuclear localization is detected.

[0011] In another embodiment of the method for assessing T cell function, the method includes detecting methylation of EOMES-373K (also referred to herein as "EOMES-373K-Me") in T cells and determining that the T cells are functional. In one example, an elevated level of EOMES-373K-Me relative to a suitable control (e.g., dysfunctional T cells) is detected in T cells. The method may also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-373K-Me in T cells. In one embodiment, the ratio of nuclear to cytoplasmic localization of EOMES-373K-Me in T cells or the ratio of cytoplasmic to nuclear localization is detected.

[0012] Another aspect of the present invention provides a method for predicting the likelihood that a subject having cancer will respond to therapy (e.g., cytotoxic therapy and / or immunotherapy), the method comprising, consisting of, or essentially consisting of the following steps: detecting post-translational modifications in the nuclear localization sequence of EOMES and / or the DNA binding motif of EOMES in T cells or T cell populations obtained from the subject, thereby predicting the likelihood that the subject will respond to therapy.

[0013] In one embodiment of the present method, the method includes detecting acetylation of EOMES-641K (also referred to herein as "EOMES-641K-Ac") in a T cell or a population of T cells to determine that the subject has an increased likelihood of resistance or non-responsiveness to therapy. In one example, the method includes detecting an increase in the level of EOMES-641K-Ac in a T cell or a population of T cells relative to a suitable control (e.g., functional T cells or T cells obtained from a healthy subject). The method may also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-641K-Ac in the T cell. In a specific example, the method includes detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Ac in the T cell or the ratio of cytoplasmic to nuclear localization.

[0014] In another embodiment of the method for predicting the likelihood of a subject with cancer responding to a therapy, the method includes detecting methylation of EOMES-641K (also referred to herein as "EOMES-641K-Me") in a T cell or a population of T cells to determine the likelihood of an increased sensitivity or responsiveness of the subject to the therapy. In one example, an elevated level of EOMES-641K-Me is detected in a T cell or a population of T cells relative to a suitable control (e.g., a dysfunctional T cell). The method may also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-641K-Me in the T cell. In one example, the ratio of nuclear to cytoplasmic localization of EOMES-641K-Me in the T cell or the ratio of cytoplasmic to nuclear localization is detected.

[0015] In another embodiment of the method for predicting the likelihood of a subject with cancer to respond to therapy, the method includes detecting methylation of EOMES-373K (also referred to herein as "EOMES-373K-Me") in a T cell or a population of T cells to determine the likelihood of an increased sensitivity or responsiveness of the subject to therapy. In one example, an elevated level of EOMES-373K-Me is detected in a T cell or a population of T cells relative to a suitable control (e.g., a dysfunctional T cell). The method may also include detecting the cellular localization (e.g., nuclear and / or cytoplasmic localization) of EOMES-373K-Me in the T cell, and optionally detecting the ratio of nuclear to cytoplasmic localization of EOMES-373K-Me in the T cell or the ratio of cytoplasmic to nuclear localization.

[0016] Another aspect of the invention relates to a method for determining the likelihood that a subject with cancer is resistant to a therapy (e.g., a cytotoxic therapy and / or an immunotherapy), the method comprising, consisting of, or consisting essentially of the following steps: detecting the presence of EOMES-641K-Ac in a T cell or T cell population obtained from the subject, thereby determining that the subject has an increased likelihood of resistance to the therapy. In one embodiment, the method comprises detecting an elevated level of EOMES-641K-Ac in the T cell or T cell population relative to a suitable control (e.g., a functional T cell or a T cell obtained from a healthy subject or a subject sensitive to a cancer therapy), indicating that the subject has an increased likelihood of resistance to the therapy. In a specific example, the method comprises contacting a sample comprising the T cell or T cell population with an antigen binding molecule that specifically binds to EOMES-641K-Ac, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Ac in the sample, thereby determining that the subject has an increased likelihood of resistance to the therapy.

[0017] Also provided herein is a method for determining the likelihood that a subject with cancer is sensitive to a therapy (e.g., cytotoxic therapy and / or immunotherapy), the method comprising, consisting of, or consisting essentially of the following steps: detecting the presence of EOMES-641K-Me in a T cell or T cell population obtained from a subject, thereby determining that the subject is more likely to be sensitive to the therapy. In one example, the method includes detecting an increase in the level of EOMES-641K-Me in a T cell or T cell population relative to a suitable control (e.g., a dysfunctional T cell or a T cell obtained from a subject resistant to cancer therapy), indicating that the subject is more likely to be sensitive to the therapy. In a specific embodiment, the method includes contacting a sample comprising a T cell or a T cell population with an antigen binding molecule that specifically binds to EOMES-641K-Me, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby determining that the subject is more likely to be sensitive to the therapy.

[0018] In another aspect, provided herein is a method for determining the likelihood that a subject with cancer is sensitive to therapy (e.g., cytotoxic therapy and / or immunotherapy), the method comprising, consisting of, or consisting essentially of the following steps: detecting the presence of EOMES-373K-Me in a T cell or T cell population obtained from the subject, thereby determining that the subject is more likely to be sensitive to therapy. In some examples, the method includes detecting an increase in the level of EOMES-373K-Me in a T cell or T cell population relative to a suitable control (e.g., a dysfunctional T cell or a T cell obtained from a subject resistant to cancer therapy), indicating that the subject is more likely to be sensitive to therapy. In a further example, the method includes contacting a sample comprising a T cell or a T cell population with an antigen binding molecule that specifically binds to EOMES-373K-Me, and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby determining that the subject is more likely to be sensitive to therapy.

[0019] In another aspect, the present invention provides a method for predicting the likelihood that a subject having cancer will respond to therapy (e.g., cytotoxic therapy and / or immunotherapy), the method comprising, consisting of, or consisting essentially of: measuring the levels of EOMES-641K-Ac and EOMES-641K-Me in T cells or T cell populations obtained from the subject; comparing the levels of EOMES-641K-Ac and EOMES-641K-Me in the T cells or T cell populations; and predicting the subject's response to therapy based on the comparison, wherein a higher level of EOMES-641K-Ac than EOMES-641K-Me indicates an increased likelihood that the subject is resistant to the therapy, and wherein a higher level of EOMES-641K-Me than EOMES-641K-Ac indicates an increased likelihood that the subject is sensitive to the therapy. In one embodiment, the method comprises contacting a sample comprising a T cell or a T cell population with a first antigen binding molecule that specifically binds to EOMES-641K-Ac and a second antigen binding molecule that specifically binds to EOMES-641K-Me; measuring the level of a first complex comprising the first antigen binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen binding molecule and EOMES-641K-Me in the sample; and predicting the likelihood of the subject's response to therapy based on the comparison, wherein a higher level of the first complex than the second complex in the sample indicates an increased likelihood that the subject is resistant to therapy, and a higher level of the second complex than the first complex in the sample indicates an increased likelihood that the subject is sensitive to therapy. The method may also involve detecting at least one other biomarker in the T cell or T cell population, such as IFN-γ, TNF-α, IL-2, Ki67, PD-1, and / or CD107a.

[0020] Also provided is a method for stratifying subjects having cancer into likely responders or non-responders to a therapy (e.g., a cytotoxic therapy and / or an immunotherapy), the method comprising, consisting of, or essentially consisting of detecting, in a sample taken from the subject, a T cell or a population of T cells comprising a post-translational modification in the nuclear localization sequence and / or DNA binding motif of EOMES, thereby stratifying the subject into a likely responder or non-responder to the therapy.

[0021] In one example, the method includes detecting EOMES-641K-Ac in a T cell or T cell population, and stratifying the subject into a possible non-responder to therapy. In a specific embodiment, the method includes contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Ac, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Ac in the sample, thereby stratifying the subject into a possible non-responder to therapy. In another embodiment, the method includes detecting EOMES-641K-Me in a T cell or T cell population, and stratifying the subject into a possible responder to therapy, for example, by contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Me, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby stratifying the subject into a possible responder to therapy. In another embodiment, the method comprises detecting EOMES-373K-Me in a T cell or T cell population and stratifying the subject into a likely responder to the therapy, for example, by contacting the sample with an antigen binding molecule that specifically binds EOMES-373K-Me and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby stratifying the subject into a likely responder to the therapy.

[0022] In one embodiment of the method for stratifying subjects having cancer into likely responders or non-responders to a therapy, the method comprises contacting a sample with a first antigen binding molecule that specifically binds to EOMES-641K-Ac and a second antigen binding molecule that specifically binds to EOMES-641K-Me; measuring in the sample the level of a first complex comprising the first antigen binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen binding molecule and EOMES-641K-Me; and stratifying the subject into a likely responder or a non-responder based on the comparison, wherein if the level of the first complex is higher than the second complex in the sample, the subject is stratified as a likely non-responder, and wherein if the level of the second complex is higher than the first complex in the sample, the subject is stratified as a likely responder.

[0023] Also provided is a method for managing a subject having cancer being treated with a therapy (e.g., a cytotoxic therapy and / or an immunotherapy), the method comprising, consisting of, or consisting essentially of the following steps: selecting a subject having cancer to be treated with the therapy based on the subject being a likely responder to the therapy, or selecting a subject having cancer not to be treated with the therapy based on the subject being a likely non-responder to the therapy, and treating the subject with or without the therapy based on this selection, wherein the selection is based on a stratification method comprising detecting T cells or T cell populations comprising a post-translational modification in a nuclear localization sequence and / or a DNA binding motif of EOMES in a sample taken from the subject, thereby stratifying the subject into a likely responder or non-responder to the therapy. In one example, the stratification method includes detecting EOMES-641K-Me in a T cell or T cell population and stratifying the subject into a possible responder to a therapy, for example, by contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Me and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby stratifying the subject into a possible responder to a therapy. In another example, the stratification method includes detecting EOMES-373K-Me in a T cell or T cell population and stratifying the subject into a possible responder to a therapy, for example, by contacting the sample with an antigen binding molecule that specifically binds to EOMES-373K-Me and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby stratifying the subject into a possible responder to a therapy. In another example, the stratification method includes detecting EOMES-641K-Ac in T cells or T cell populations and stratifying patients as likely non-responders to therapy, for example, by contacting the sample with an antigen-binding molecule that specifically binds to EOMES-641K-Ac and detecting a complex comprising the antigen-binding molecule and EOMES-641K-Ac in the sample, thereby stratifying patients as likely non-responders to therapy. In a specific embodiment, the stratification method comprises contacting a sample with a first antigen binding molecule that specifically binds to EOMES-641K-Ac and a second antigen binding molecule that specifically binds to EOMES-641K-Me; measuring the level of a first complex comprising the first antigen binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen binding molecule and EOMES-641K-Me in the sample; and stratifying the subject into a possible responder or a non-responder based on the comparison, wherein if the level of the first complex is higher than the second complex in the sample, the subject is stratified as a possible non-responder, and wherein if the level of the second complex is higher than the first complex in the sample, the subject is stratified as a possible responder.

[0024] In some examples of the above methods, the method further comprises detecting at least one other biomarker in the T cell or T cell population, such as IFN-γ, TNF-α, IL-2, Ki67, PD-1 and / or CD107a.

[0025] In yet another aspect, provided is a method for assessing immune function in a subject, the method comprising, consisting of, or consisting essentially of detecting post-translational modifications in the nuclear localization sequence of EOMES and / or the DNA binding motif of EOMES in T cells or a population of T cells obtained from the subject.

[0026] In one embodiment, the method involves detecting acetylation of EOMES-641K (also referred to herein as "EOMES-641K-Ac") in a T cell or a population of T cells to determine that the subject's immune function is impaired. For example, the method can include detecting an elevated level of EOMES-641K-Ac in a T cell or a population of T cells relative to a suitable control (e.g., a T cell obtained from a subject with normal or active immune function). In some embodiments, the cellular localization of EOMES-641K-Ac in the T cell is detected, such as nuclear and / or cytoplasmic localization. In a specific example, the method includes detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Ac in the T cell or the ratio of cytoplasmic to nuclear localization.

[0027] In another embodiment, the method for assessing immune function includes detecting methylation of EOMES-641K (also referred to herein as "EOMES-641K-Me") in a T cell or a T cell population to determine that the subject has normal or active immune function, such as detecting an elevated level of EOMES-641K-Me in a T cell or a T cell population relative to a suitable control (e.g., T cells from an immunocompromised subject). In one example, the cellular localization of EOMES-641K-Me in T cells, such as nuclear and / or cytoplasmic localization, is detected. In a specific example, the method includes detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Me in T cells or the ratio of cytoplasmic to nuclear localization.

[0028] In another embodiment, the method comprises detecting methylation of EOMES-373K (also referred to herein as "EOMES-373K-Me") in a T cell or a population of T cells to determine that the subject has normal or active immune function, for example, detecting elevated levels of EOMES-373K-Me in a T cell or a population of T cells relative to a suitable control (e.g., T cells from an immunocompromised subject). In one embodiment, the method comprises detecting the cellular localization of EOMES-373K-Me in a T cell, for example, detecting nuclear and / or cytoplasmic localization of EOMES-373K-Me in a T cell, and optionally detecting the ratio of nuclear to cytoplasmic localization of EOMES-373K-Me in a T cell or a ratio of cytoplasmic to nuclear localization.

[0029] Also provided is an antigen binding molecule that specifically binds to EOMES-641K-Ac, which is suitable for assessing T cell function, for predicting the likelihood of a subject with cancer to respond to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is resistant to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is sensitive to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for stratifying subjects with cancer into likely responders or non-responders to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for managing the treatment of subjects with cancer with a therapy (e.g., cytotoxic therapy and / or immunotherapy), for assessing the immune function of a subject and / or managing the treatment of subjects with impaired or decreased immune function with a therapy (e.g., immunotherapy).

[0030] In another aspect, a complex is provided, comprising EOMES-641K-Ac and an antigen binding molecule that specifically binds to EOMES-641K-Ac.

[0031] In another aspect, an antigen binding molecule that specifically binds to EOMES-641K-Me is provided, which is suitable for assessing T cell function, for predicting the likelihood of a subject with cancer to respond to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is resistant to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is sensitive to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for stratifying subjects with cancer into likely responders or non-responders to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for managing the treatment of subjects with cancer with a therapy (e.g., cytotoxic therapy and / or immunotherapy), for assessing the immune function of a subject and / or managing the treatment of a subject with impaired or decreased immune function with a therapy (e.g., immunotherapy).

[0032] Also described herein is a complex comprising EOMES-641K-Me and an antigen binding molecule that specifically binds to EOMES-641K-Me.

[0033] In another aspect, an antigen binding molecule that specifically binds to EOMES-373K-Me is provided, which is suitable for assessing T cell function, for predicting the likelihood of a subject with cancer to respond to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is resistant to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is sensitive to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for stratifying subjects with cancer into likely responders or non-responders to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for managing the treatment of subjects with cancer with a therapy (e.g., cytotoxic therapy and / or immunotherapy), for assessing the immune function of a subject and / or managing the treatment of a subject with impaired or decreased immune function with a therapy (e.g., immunotherapy).

[0034] Also provided is a complex comprising EOMES-373K-Me and an antigen-binding molecule that specifically binds to EOMES-373K-Me.

[0035] Another aspect of the present invention provides a kit for assessing T cell function, for predicting the likelihood of a subject with cancer to respond to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is resistant to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for determining the likelihood that a subject with cancer is sensitive to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for stratifying subjects with cancer into likely responders or non-responders to a therapy (e.g., cytotoxic therapy and / or immunotherapy), for managing the treatment of a subject with cancer with a therapy (e.g., cytotoxic therapy and / or immunotherapy), for assessing the immune function of a subject and / or managing the treatment of a subject with impaired or decreased immune function with a therapy (e.g., immunotherapy), wherein the kit comprises at least one, at least two, or each of an antigen-binding molecule that specifically binds to EOMES-641K-Ac, an antigen-binding molecule that specifically binds to EOMES-641K-Me, and an antigen-binding molecule that specifically binds to EOMES-373K-Me. In one embodiment, the kit further comprises one or more controls, including a positive control and a negative control, for example, wherein the positive control is selected from EOMES-641K-Ac polypeptide, EOMES-641K-Me polypeptide, and EOMES-373K-Me polypeptide. The kit may optionally include instructional materials for performing the methods described above and herein.

[0036] Another aspect of the present invention relates to a T cell comprising a complex comprising EOMES-641K-Ac and a first antigen binding molecule that specifically binds to EOMES-641K-Ac; EOMES-641K-Me and a first antigen binding molecule that specifically binds to EOMES-641K-Me; or EOMES-373-Me and a first antigen binding molecule that specifically binds to EOMES-373K-Me. In one example, the T cell further comprises a second antigen binding molecule that binds to the first antigen binding molecule, for example, a second antigen binding molecule that comprises a detectable label.

[0037] In one embodiment of the methods, kits, antigen binding molecules, complexes or T cells described above and herein, therapy is immunotherapy, such as immune checkpoint inhibitors. Exemplary inhibitors include antagonist antigen binding molecules (e.g., antibodies) that specifically bind to immune checkpoint molecules. In one example, antagonist antigen binding molecules (e.g., antibodies) specifically bind to immune checkpoint molecules selected from PD-1, PD-L1 and CTLA4. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Schematic diagrams and images showing the isolated CD8 + Prevalence of EOMES in T cells. CD8 T cells were isolated from liquid biopsies of healthy donors (HD), patients with metastatic breast cancer, or patients with melanoma every 3 months for 24 months after baseline blood collection. + T cells. According to the objective response to immunotherapy treatment (using single or dual therapy of pembrolizumab, nivolumab and / or ipilimumab), melanoma patients are further divided into complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). The plotted graph shows the tumor growth change % for PD (progressive disease) or CR (complete response) patient cohorts as described in RECIST 1.1. (A) As described above, CD8 + T cells. Cells were stimulated with phorbol-12-myristate 13-acetate combined with calcium ionophore A23187 (PMA / CI) or not, and then fixed. + T cells were immunostained with anti-Ki67 primary antibody for NS or ST CD8 + T cells were stained with anti-TNF-α or anti-IFN-γ primary antibodies and DAPI for immunofluorescence microscopy. The figures represent the nuclear fluorescence intensity (NFI) values ​​of Ki67 and the total fluorescence intensity (TFI) values ​​of TNF-α and IFN-γ, and were measured using ImageJ to select nuclei and subtract background (n>20 individual cells per patient for a cohort of 10 patients). (B) CD8 T cells were isolated from healthy donors (HD) and melanoma patients classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) as described above. +T cells. The cells were fixed and immunofluorescence microscopy was performed using a primary antibody for EOMES and PD1 together with DAPI staining. These figures represent the NFI value of the EOMES complex and the average TFI of PD-1, and were measured using a universal scanning and analysis system (Applied Scientific Instrumentation; ASI), which performs high-throughput IF microscopy on multiplexed immunofluorescence samples to quantify cell number and IF signal intensity (for a cohort of 10 patients, n>2500 individual cells per patient). The figure represents % cell population. (C) EOMES protein structure, which shows the NLS domain. (D) Schematic diagram of EOMES plasmids: EOMES WT (E-WT; wild-type EOMES); EOMES mutant 1 (E-MUT1; EOMES with an alanine mutation at lysine 641, which mimics the non-acetylated or non-methylated state of lysine residues); and EOMES mutant 2 (E-MUT2; EOMES with a phenylalanine mutation at lysine 641, which mimics the hypermethylated state of lysine residues). (E) Jurkat T cells were transfected with empty vector (VO), E-WT, E-MUT1, or E-MUT2 alone and probed with anti-EOMES, anti-TBET, and anti-PD1 antibodies. The plots represent the Fn / c of ​​EOMES, the average NFI of EOMES, or the average TFI of PD-1, measured using ImageJ with background subtraction (n=>20 cells for each group). Representative images of each data set are shown. The scale bar is shown in orange and is equivalent to 10 mm in length. (F) Jurkat T cells transfected with VO, E-WT, E-MUT1, or E-MUT2 were probed with anti-Ki67, anti-IFN-γ, and anti-TNF-α antibodies. Graphs show the TFI of Ki67, IFN-γ, and TNF-α measured using ImageJ with background subtraction (n=>20 cells for each group).

[0039] Figure 2 Graphical representation of the specificity of polyclonal rabbit antibodies raised against various EOMES polypeptides. (A) Antibody raised against an EOMES polypeptide without post-translational modifications. (B) Antibody raised against an EOMES polypeptide acetylated at lysine 641. (C) Antibody raised against an EOMES polypeptide methylated at lysine 641. (D) Antibody raised against an EOMES polypeptide methylated at lysine 373.

[0040] Figure 3Schematic diagram and graphic representation, it shows the ability of anti-EOMES antibodies to predict patients' responses to immunotherapy. (A) EOMES protein structure, it shows the DNA binding domain. (B) Schematic diagram of the generation of polyclonal antibodies specific for NLS motifs or DNA binding motifs, these motifs are unmodified or modified by methylation (Me) or acetylation (Ac). (C) Using BOND RX (Leica Biosystems), formalin-fixed paraffin-embedded (FFPE) samples (ultimately divided into responders or resistance cohorts) of melanoma patients from primary tumor baseline biopsies are processed for 3D high-resolution microscopy. FFPE tissues are fixed and immunofluorescence microscopy is performed by using a primary antibody for CD8, acetylated EOMES (anti-EOMES-641K-Ac; "EOMES-Ac") or methylated EOMES (anti-EOMES-641K-Me; "EOMES-Me") together with DAPI staining to detect the samples. The plots show the mean NFI values ​​for EOMES-Me and the mean NFI for EOMES-Ac, measured using ImageJ with background subtraction (N = 90 cells for each patient sample. N = 10 patients per cohort). (D) Liquid biopsies were obtained from consenting melanoma patients every 3 months for 24 months after baseline blood draw. Patients were further divided into those resistant or responding to immunotherapy based on their objective response to treatment with immunotherapy (single or dual therapy with pembrolizumab, nivolumab, and / or ipilimumab). CD8 isolated from the blood of the resistant cohort or responder cohort defined according to RECIST 1.1 was detected using anti-CD8 antibody and anti-EOMES-641K-Ac polyclonal antibody. + T cells were screened. The cells were fixed and stained with antibodies and DAPI for immunofluorescence microscopy. The plots show the mean NFI and cytoplasmic fluorescence intensity (CFI) of EOMES-641K-Ac measured using ImageJ with background subtraction, N = 10 patients per cohort (n = 10 patients per group). (E) Liquid biopsies were obtained from consenting melanoma patients every 3 months for 24 months after baseline blood draw. Patients were further divided into those resistant or responding to immunotherapy based on their objective response to immunotherapy treatment (single or dual therapy with pembrolizumab, nivolumab and / or ipilimumab). CD8 isolated from the blood of the resister cohort or responder cohort defined according to RECIST 1.1 was detected using anti-CD8 antibodies and anti-EOMES-641K-Me polyclonal antibodies. +T cells were screened. The cells were fixed and stained with antibodies and DAPI for immunofluorescence microscopy. The plots show the average NFI and CFI of EOMES-641K-Me measured using ImageJ with background subtraction (n = 10 patients per group). (F) Liquid biopsies were taken from consenting melanoma patients every 3 months within 24 months after baseline blood collection. Based on the objective response to immunotherapy treatment (single or dual therapy with pembrolizumab, nivolumab and / or ipilimumab), patients were further divided into those resistant to or responding to immunotherapy. CD8 isolated from the blood of the resistant cohort or responder cohort defined according to RECIST 1.1 was detected using anti-CD8 antibodies and anti-EOMES-373K-Me polyclonal antibodies. + T cells were screened. The cells were fixed and stained with antibodies and DAPI for immunofluorescence microscopy. The plots show the average NFI and CFI of EOMES-373K-Me measured using ImageJ with background subtraction (n = 10 patients per group). (G) Liquid biopsies were taken every month from informed consenting patients with BRACA-positive, triple-negative breast cancer (TNBC) within 12 months after baseline blood collection. CD8 isolated from the blood of the resistant cohort or responder cohort defined according to RECIST 1.1 was detected using anti-CD8 antibody, anti-EOMES-641K-Me polyclonal antibody or anti-EOMES-641K-Ac polyclonal antibody. + T cells were screened. Cells were fixed and stained with antibodies and DAPI for immunofluorescence microscopy. The graphs show the mean FI of EOMES-641K-Ac and FI of EOMES-641K-Me measured using ImageJ with background subtraction (n = 10 patients per group).

[0041] Figure 4 Figure 2 is a graphical representation showing the prevalence of post-translationally modified EOMES in brain cancer metastases. Primary tumor brain FFPE sections from 10 patient samples were processed using BOND RX and OPAL-5 multicolor automation kits (from Perkin Elmer). FFPE tissues were fixed and stained with anti-EOMES-641K-Ac, anti-EOMES-641K-Me, anti-cytokeratin (a tumor marker) and anti-CD8 antibodies and DAPI (green=EOMES-641K-Ac, cyan=EOMES-EOMES-641K-Me, red=CD8, magenta=cytokeratin, DAPI=blue), along with Opal kit dyes 520, 570, 650 and 690 for immunofluorescence microscopy. CD8 was measured. +and EOMES-641K-Ac or EOMES-641K-Me positive CD8 + T cell population %. (A) The graph shows EOMES-641K-Ac or EOMES-641K-Me positive CD8 + T cell population%. (B) The graph shows the CD8 + TFI of EOMES-641K-Ac and EOMES-641K-Me in T cells (+ / - standard error). (N = 10 patients, N of at least 50,000 cells were analyzed for each patient's FFPE sample). DETAILED DESCRIPTION

[0042] 1. Definition

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods or materials similar or equivalent to those described herein can be used to implement or test the present invention, preferred methods or materials are described. For the purposes of the present invention, the following terms are defined as follows.

[0044] As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0045] The term "about" as used herein refers to the usual error range of the corresponding numerical value that is readily known to those skilled in the art. The "about" numerical value or parameter mentioned herein includes (and describes) embodiments involving the numerical value or parameter itself.

[0046] The terms "simultaneous administration..." or "administered simultaneously..." or "co-administered," etc., refer to the administration of a single composition containing two or more actives, or the administration of each active in separate compositions and / or by separate routes of delivery, either concurrently or simultaneously or sequentially within a sufficiently short period of time to provide an effective result equivalent to that obtained when all such actives are administered as a single composition. By "contemporaneously" is meant that the active agents are administered at substantially the same time, and desirably are administered together in the same formulation. By "contemporaneously" is meant that the active agents are administered in close time, for example, one agent is administered within about 1 minute to about 1 day before or after another agent. Any contemporaneous time is acceptable. However, it will often be the case that, when not administered concurrently, the agents are administered within about 1 minute to about 8 hours, and suitably within less than about 1 to about 4 hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes a precise location, but can also be within a range of about 0.5 to about 15 centimeters, preferably within a range of about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at regular intervals, for example, at intervals of about 1 day to several weeks or months. The active agents can be administered in any order. The term "sequentially" as used herein means that the agents are administered in sequence, for example, at intervals of several minutes, hours, days or weeks. If appropriate, the active agents can be administered in regularly repeating cycles.

[0047] The term "agent" refers to any diagnostic, therapeutic or preventive agent. The term "agent" should not be understood in a narrow sense, but should be extended to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins, and genetic molecules such as RNA, DNA and their mimetics and chemical analogs, and cellular agents. The term "agent" includes cells that can produce and secrete the polypeptides referred to herein and the polynucleotides comprising the nucleotide sequence encoding the polypeptides. The term "agent" also extends to nucleic acid constructs, including vectors such as viral or non-viral vectors, expression vectors and plasmids, for expression and secretion in a range of cells.

[0048] As used herein, "amplification" generally refers to the process of producing multiple copies of a desired sequence. "Multiple copies" means at least two copies. "Copy" does not necessarily mean perfect sequence complementarity or identity with the template sequence. For example, copies can include nucleotide analogs, such as deoxyinosine, intentional sequence changes (e.g., sequence changes introduced by primers containing sequences that hybridize to but are not complementary to the template), and / or sequence errors that occur during the amplification process.

[0049] The "amount" or "level" of a biomarker is the level or amount detectable in a sample. These can be measured by methods known to those skilled in the art and also disclosed herein. These terms include quantitative amounts or levels (e.g., weight or molar), semi-quantitative amounts or levels, relative amounts or levels (e.g., weight % or molar % within a class), concentrations, etc. Thus, these terms include absolute or relative amounts or levels or concentrations of a biomarker in a sample. The expression level or amount of the assessed biomarker can be used to determine the response to treatment.

[0050] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative (or).

[0051] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule (such as a receptor).

[0052] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of the target. In some embodiments, the term can refer to an antibody that prevents the target to which it binds (e.g., PD-1, CTLA4, etc.) from exerting its biological function.

[0053] As used herein, "anti-immune checkpoint molecule antagonist antibody" refers to an antibody that can inhibit the biological activity and / or downstream events (one or more) mediated by immune checkpoint molecules. Anti-immune checkpoint molecule antagonist antibodies cover antibodies that block, antagonize, suppress or reduce (to any extent, including significantly) the biological activity of immune checkpoint molecules, wherein the biological activity of immune checkpoint molecules includes inhibitory signal transduction through immune checkpoint molecules and downstream events mediated by immune checkpoint molecules, such as the binding of immune checkpoint molecule binding partners to immune checkpoint molecules and downstream signal transduction, inhibition of cell proliferation (including tumor proliferation), inhibition of T cell proliferation, inhibition of T cell activation, inhibition of cytokine secretion and inhibition of anti-tumor immune response. For the purposes of the present invention, it should be clearly understood that the term "anti-immune checkpoint molecule antagonist antibody" (interchangeably referred to as "antagonist immune checkpoint molecule antibody", "antagonist anti-immune checkpoint molecule antibody" or "immune checkpoint molecule antagonist antibody") covers all previously identified terms, names, and functional states and characteristics, in which the immune checkpoint molecule itself, the biological activity of the immune checkpoint molecule, or the results of the biological activity are substantially abolished, reduced or neutralized to any meaningful extent.

[0054] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies) and single variable domain antibodies, so long as they exhibit the desired biological activity. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR or V H ) and heavy chain constant region. The heavy chain constant region contains three domains, namely C H1 、C H2 and C H3 Each light chain comprises a light chain variable region (which may be abbreviated as LCVR or V L ) and the light chain constant region. The light chain constant region contains one domain (C L1 ). V H and V L The regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), V H and V L Interspersed within the region are more conserved regions, called framework regions (FR). H and V L It consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the antibody (or its antigen-binding portion) may be identical to human germline sequences, or may be natural or artificially modified. Based on a side-by-side analysis of two or more CDRs, an amino acid consensus sequence may be defined. Included within the term "antibody" are antibodies of any class, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not necessarily belong to any particular class. Immunoglobulins can be divided into different classes based on the antibody amino acid sequence of the constant region of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0055] "Antigen binding fragment" can be provided by arranging one or more CDRs on a non-antibody protein scaffold. "Protein scaffold" as used herein, includes but is not limited to immunoglobulin (Ig) scaffolds, such as IgG scaffolds, which can be four-chain or two-chain antibodies, or they can only contain the Fc region of an antibody, or they can contain one or more constant regions from an antibody, which can be of human or primate origin, or they can be artificial chimeras of human and primate constant regions. The protein scaffold can be an Ig scaffold, such as an IgG or IgA scaffold. The IgG scaffold can contain some or all of the domains of an antibody (i.e., CH1, CH2, CH3, V H 、V L ). The antigen binding protein may comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgG1. The scaffold may consist of, comprise, or be a portion of the Fc region of an antibody. Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark-derived variable IgNAR domains are also included in the expression "antigen-binding fragment" as used herein. The antigen-binding fragment of an antibody generally comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR that is adjacent to or in frame with one or more framework sequences. In the case of a fragment having a CDR that is adjacent to or in frame with one or more framework sequences, the CDR is preferably a CDR that is adjacent to or in frame with one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be dimerized and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V LIn certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H1 (ii) V H -C H2 (iii) V H -C H3 (iv) V H -C H1 -C H 2; (v) V H -C H1 -C H2 -C H3 ; (vi) V H -C H2 -C H3 ; (vii) V H -C L ;(viii)V L -C H 1; (ix) V L -C H2 ;(x)V L -C H3 ;(xi)V L -C H1 -C H2 ;(xii)V L -C H1 -C H2 -C H3 ;(xiii)V L -C H2 -C H3 ; and (xiv) V L -C L . In any configuration of variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or may be connected through all or part of a hinge region or a linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment of the antibody of the present invention may comprise homodimers or heterodimers (or other multimers) of any variable and constant domain configuration listed above, which are non-covalently associated with each other and / or with one or more monomer V H or V LThe antigen-binding fragments of an antibody are formed by binding to a plurality of domains of the antibody. ... As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of an antibody generally comprise at least two different variable domains, each of which is capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antigen-binding molecule format, including the exemplary bispecific antigen-binding molecule formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of the antibodies of the invention using conventional techniques available in the art.

[0056] As used herein, the term "antigen" and its grammatical equivalents (e.g., "antigenic") refer to a compound, composition, or substance that can be specifically bound by a specific humoral or cellular immune product (e.g., an antibody molecule or a T-cell receptor). Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, carbohydrates (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common antigen classifications include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoan and other parasitic antigens, tumor antigens, antigens involved in autoimmune diseases, allergies, and transplant rejection, toxins, and other miscellaneous antigens.

[0057] By "antigen binding molecule" is meant a molecule that has binding affinity for a target antigen. It should be understood that the term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin-derived protein frameworks that exhibit antigen-binding activity. Representative antigen-binding molecules that can be used to practice the present invention include antibodies and antigen-binding fragments.

[0058] The term "antigen presenting cell" or "APC" refers to a cell that is capable of presenting one or more antigens in the form of peptide-MHC complexes that can be recognized by specific effector cells of the immune system (also referred to herein as "immune effector cells") and thereby modulate (e.g., stimulate / enhance or reduce / tolerate / abort) the immune response to the presented one or more antigens. In certain embodiments of the present invention, APCs are capable of activating immune effector cells, such as T lymphocytes, including CD8 + and / or CD4 + Lymphocytes. Cells with the potential to act as APCs in the body include not only professional APCs, such as dendritic cells, macrophages, Langerhans cells, monocytes, and B cells, but also non-professional APCs, illustrative examples of which include activated epithelial cells, fibroblasts, glial cells, pancreatic β cells, and vascular endothelial cells, as well as cancer cells. Many types of cells can present antigens on their cell surfaces for recognition by immune effector cells (including T cells).

[0059] As used herein, the term "antigen specificity" refers to a property of a cell population such that presentation of a particular antigen or antigen fragment results in specific cell proliferation, suitably T cell proliferation characterized by, for example, activation of T cells (e.g., CTLs and / or helper T cells), such T cell activation being suitably directed against damaged cells, malignancies, or infections.

[0060] As used herein, the terms "binding," "specific binding," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to the target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0061] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic, and / or prognostic indicator. The biomarker can be used as an indicator of a specific subtype of a disease or condition (such as cancer), characterized by specific, molecular, pathological, histological, and / or clinical characteristics, and / or can be used as an indicator of a specific cell type or state (such as epithelial cells, mesenchymal cells, etc.) and / or response to therapy. Biomarkers include but are not limited to polynucleotides (such as DNA and / or RNA), polynucleotide copy number changes (such as DNA copy number), polypeptides, polypeptides, and polynucleotide modifications (such as post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers. Biomarkers can be present in samples obtained from subjects before the onset of physiological or pathophysiological states (such as primary cancer, metastatic cancer, etc.), including their symptoms (such as, response to therapy). Therefore, the presence of biomarkers in samples obtained from subjects can indicate that the risk of forming a physiological or pathophysiological state or its symptoms increases. Alternatively or additionally, the biomarker may be normally expressed in an individual, but its expression may change (i.e., it increases (up-regulated; over-expressed) or decreases (down-regulated; under-expressed)) before the onset of a physiological or pathophysiological state (including its symptoms). Thus, a change in the level of the biomarker can indicate that the subject is at increased risk of developing a physiological or pathophysiological state or its symptoms. Alternatively or additionally, a change in the level of a biomarker can reflect a change in a particular physiological or pathophysiological state or its symptoms in a subject, thereby allowing the nature (e.g., severity) of the physiological or pathophysiological state or its symptoms to be tracked over a period of time. This approach can be used, for example, to monitor a treatment regimen to assess its effectiveness (or other aspects) in a subject. As described herein, reference to the level of a biomarker includes the concentration of the biomarker, the expression level of the biomarker, or the activity of the biomarker.

[0062] The terms "biomarker signature," "signature," "biomarker expression signature," or "expression signature" are used interchangeably herein and refer to a biomarker or combination of biomarkers whose expression is an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator. A biomarker signature can be an indicator of a particular subtype of a disease or condition (e.g., primary cancer, metastatic cancer, etc.) or its symptoms (e.g., response to therapy, drug resistance, and / or disease burden), characterized by specific molecular, pathological, histological, and / or clinical characteristics. In some embodiments, the biomarker signature is a "gene signature." The term "gene signature" can be used interchangeably with "gene expression signature" and refers to a polynucleotide or combination of polynucleotides whose expression is an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator. In some embodiments, the biomarker signature is a "protein signature." The term "protein signature" can be used interchangeably with "protein expression signature" and refers to a polypeptide or combination of polypeptides whose expression is an indicator, e.g., a predictive, diagnostic, and / or prognostic indicator. A biomarker signature may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more biomarkers.

[0063] The terms "cancer" and "cancerous" refer to or describe the physiological condition in a subject that is typically characterized by uncontrolled cell growth with the potential to invade locally and / or spread to other parts of the body (metastasis). The term "cancer" herein is generally used interchangeably with "tumor" (unless a tumor is specifically referred to as a "benign" tumor, i.e., an abnormal mass of cells that lacks the ability to invade adjacent tissues or metastasize), and includes malignant solid tumors (e.g., carcinomas, sarcomas) and malignant growths in which no solid tumor mass may be detectable (e.g., certain hematologic malignancies). Non-limiting examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal cancer, carcinoma, penile cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia ( Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with leukomatosis, edema (such as that associated with brain tumors), Meig's syndrome, brain cancer, and head and neck cancer, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid cancer, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. In still some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers. In particular embodiments, the cancer is melanoma or lung cancer, suitably metastatic melanoma or metastatic lung cancer.

[0064] The term "cellular compartment" includes parts of a cell, including organelles (eg, mitochondria, Golgi apparatus, endoplasmic reticulum, ribosomes, etc.), nucleus, cytoplasm (optionally including organelles), nuclear membrane, cell membrane, and other cellular regions.

[0065] The term "chemotherapy" refers to treatment of humans or animals with one or more chemotherapeutic agents that inhibit or eliminate cell growth and cell division, i.e., the treatment is used as a cell proliferation inhibitor or to induce cell death (apoptosis). Cancer cells have uncontrolled growth and division compared to normal cells, so chemotherapy should be more effective against cancer cells.

[0066] The term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include erlotinib ( Genentech / OSI Pharm.), bortezomib ( Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant ( AstraZeneca), sunitinib ( Pfizer / Sugen), letrozole ( Novartis), imatinib mesylate ( Novartis), finasunate ( Novartis), oxaliplatin ( Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, Wyeth), lapatinib ( GSK572016, Glaxo Smith Kline), lonafarnib (SCH 66336), sorafenib ( Bayer Labs), gefitinib ( AstraZeneca), AG1478, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethylenimine and methylamelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; annonaceous lactones (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan) bryostatin; callystatin; CC-1065 (including its synthetic analogs adolesin, callylesin, and biszelotin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talc duocarmycin, including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, naphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolidinone hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma and calicheamicin omega (Angew Chem. Intl. Ed. Engl.1994 33:183-186); daptomycins, including daptomycin A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related pigment protein enediyne antibiotic chromophores), aclacinomycins, actinomycins, authramycins, azoserine, bleomycin, actinomycin C, carrubicin, carmomycin, chromomycin, dactinomycin, daunorubicin, detoximum iodide, 6-diazo-5-oxo-L-norleucine, . (doxorubicin), morpholinyl doxorubicin, cyanomorpholinyl doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin, streptozocin, streptozotocin, tubercidin in), ubenimex, zoloft, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; and androgens antiadrenergic agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; busamustine; bisantrene; idatrexate; defosfamide; colcemid; diazocone; ilonicid; elliptonium acetate; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidamnol; niterazol; pentostatin; methamine mustard; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizolan; spirogermanium; tenuazonic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculocin A, and serpentin); urethane; vindesine; dacarbazine; mannitol mustard; mitobronitol; dibromodulcitol; propidium bromide; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), (without Cremophor), an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and (docetaxel, docetaxel; Sanofi-Aventis); chlorambucil; (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; mitoxantrone (novantrone); teniposide; idatrexate; daunorubicin; aminopterin; capecitabine Ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0067] Chemotherapeutic agents also include (i) antihormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, troloxifene, keoxifene, LY117018, onapristone, and (toremifene citrate); (ii) aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, (Megestrol acetate), (exemestane; Pfizer), formestane, fadrozole, (Voroxazole), (letrozole; Novartis) and (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation (e.g., PKC-α, Ralf, and H-Ras); (vii) ribozymes, such as inhibitors of VEGF expression (e.g., ) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, such as and rIL-2; topoisomerase 1 inhibitors, such as rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0068] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab ( Genentech); Cetuximab ( Imclone); Panitumumab ( Amgen), rituximab ( Genentech / Biogen Idec), Pertuzumab ( 2C4, Genentech), trastuzumab ( Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin ( Other humanized monoclonal antibodies with therapeutic potential as pharmaceutical agents in combination with the compounds of the present invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol), cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin), ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, and pefucituzumab pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumabTetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant, fully human, full-length IgG1λ antibody genetically modified to recognize the interleukin-12p40 protein.

[0069] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, and may alternatively be referred to as "EGFR antagonists." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCCCRL HB8507), MAb 225 (ATCCCRL 8508), MAb 528 (ATCCCRL 8509) (see U.S. Pat. No. 4,943,533 to Mendelsohn et al.), and variants thereof, such as chimeric 225 (C225 or cetuximab; ) and remodeled human 225 (H225) (see, WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR-targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized antibodies and chimeric antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody to EGFR that competes with EGF and TGF-α for EGFR binding (EMD / Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, and described in U.S. Patent No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents to produce immunoconjugates (e.g., see EP 659439 A2, Merck Patent GmbH). EGFR antagonists include small molecules such as those described in: Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,14 Nos. 0,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037. Specific small molecule EGFR antagonists include OSI-774 (CP-358774), erlotinib, Genentech / OSI Pharmaceuticals); PD183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib 4-(3'-chloro-4'-fluoroaniline)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenylamino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); EGFR / HER2 dual tyrosine kinase inhibitors, such as lapatinib ( GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

[0070] Chemotherapeutic agents also include "tyrosine kinase inhibitors," which include the EGFR-targeted drugs mentioned in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 provided by Takeda; CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); HER dual inhibitors, such as EKB-569 (provided by Wyeth), which preferentially binds to EGFR but simultaneously inhibits cells overexpressing both HER2 and EGFR; lapatinib (GSK572016; provided by Glaxo-SmithKline), an oral inhibitor of HER2 and EGFR tyrosine kinases; PKI-166 (provided by Novartis); broad-spectrum HER inhibitors, such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors, such as the antisense agent ISIS-5132 provided by ISIS Pharmaceuticals, which inhibits Raf-1 signaling; non-HER-targeted TK inhibitors, such as imatinib mesylate ( provided by Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib ( VEGF receptor tyrosine kinase inhibitors, such as vatalanib (PTK787 / ZK222584, provided by Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (provided by Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); broad-spectrum HER inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS); 3521; Isis / Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, ); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO 1999 / 06396 (Warner Lambert); WO 1996 / 30347 (Pfizer, Inc); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca) and WO 1996 / 33980 (Zeneca).

[0071] Chemotherapeutic agents also include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, and selenomethorphan. acetate), ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium sodium), quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronic acid and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0072] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, and daptomycin. dipropionate), betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone hexanoate, fluocortolone pivalate, and flupredniidine acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN Bio Therapeutics, LLC); antirheumatic drugs, such as azathioprine, cyclosporine (cyclosporin A), D-penicillamine, gold salts (gold salts), hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNF-α) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab Interleukin-13 (IL-13) blockers such as lebrikizumab; interferon-alpha (IFN) blockers such as rontalizumab; β7 integrin blockers such as rhuMAb β7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin α (LTa); radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioisotopes of Lu); miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate / phenylbutyrate, ET-18-OCH3 or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, proanthocyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur Bexarotene Bisphosphonates such as clodronate (e.g. or ), etidronate NE-58095, Zoledronic Acid / Zoledronate Alendronate Pamidronate tiludronate or risedronate and epidermal growth factor receptor (EGF-R); vaccines e.g. Vaccines; perifosine, COX-2 inhibitors (eg, celecoxib or etoricoxib), proteosome inhibitors (eg, PS341); CCI-779; tipifarnib (R11577); sorafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium Pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR TM ); and pharmaceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, which is an abbreviation for combination therapy with oxaliplatin (ELOXATIN TM ) is an abbreviation for the combined treatment regimen of 5-FU and leucovorin.

[0073] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. The specific example of NSAIDs includes aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen (ketoprofen), flurbiprofen, oxaprozin (oxaprozin) and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam (droxicam), lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib (rofecoxib) and valdecoxib (valdecoxib). NSAIDs are indicated for the relief of symptoms of diseases such as rheumatoid arthritis, osteoarthritis, inflammatory joint diseases, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0074] The term "clinical outcome" or "clinical endpoint" refers to any clinical observation or measurement related to a patient's response to therapy. Non-limiting examples of clinical outcomes include tumor response (TR), overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), time before tumor recurrence (TTR), time before tumor progression (TTP), relative risk (RR), toxicity, or side effects. "Overall survival" (OS) means an extension of life expectancy compared to an individual or patient in a natural state or who has not received treatment. "Progression-free survival" (PFS) or "time before tumor progression" (TTP) indicates the length of time that cancer does not grow during and after treatment. Progression-free survival includes the amount of time a patient experiences a complete response or partial response, as well as the amount of time a patient experiences stable disease. "Tumor recurrence," as used herein and as defined by the National Cancer Institute, means that cancer has returned (come back), usually after a period of time in which the cancer cannot be detected. Cancer may reappear in the same location as the original (primary) tumor, or in other parts of the body. It is also known as recurrent cancer. "Time to recurrence" (TTR) is defined as the time from the date of cancer diagnosis to the first recurrence, death, or the last contact (if the patient did not have any recurrence of cancer at the time of last contact). If the patient did not relapse, the TTR was censored at the time of death or last follow-up. "Relative risk" (RR), in statistics and mathematical epidemiology, refers to the risk of an event (or developing a disease) associated with an exposure. The relative risk is the ratio of the probability of the event occurring in the exposed group to the probability of the event occurring in the unexposed group.

[0075] As used herein, the term "complex" refers to a combination or aggregate formed by molecules (e.g., peptides, polypeptides, etc.) in direct and / or indirect contact with each other. In specific embodiments, "contact", or more specifically, "direct contact" means that two or more molecules are close enough to each other so that attractive non-covalent interactions (e.g., van der Waals forces, hydrogen bonding, ionic and hydrophobic interactions, etc.) dominate the interaction of molecules. In such embodiments, the complex of molecules (e.g., peptides and polypeptides) is formed under such conditions that the complex is thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed state of its constituent molecules). As used herein, the term "polypeptide complex" or "protein complex" refers to a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, elevenmer, twelvemer or higher order oligomers. In specific embodiments, the polypeptide complex is formed by the combination of EOMES with an antigen binding molecule specific for EOMES.

[0076] Throughout this specification, unless the context requires otherwise, the words "comprise," "include," and "contain" will be understood to mean the inclusion of a specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Thus, the term "comprising" and similar usages indicate that the listed elements are required or mandatory, but other elements are optional and may or may not be present. By "consisting of" is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. By "consisting essentially of" is meant to include any element listed after the phrase, and be limited to other elements that do not interfere with or promote the activity or behavior of the listed elements specified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or behavior of the listed elements.

[0077] As used herein, the term "correlation" or "associated with..." and similar terms refer to a statistical association between two or more things (e.g., events, features, results, numbers, data sets, etc., which may be referred to as "variables"). It should be understood that these things can be of different types. Variables are typically represented as numbers (e.g., measurements, values, probabilities, risks), where a positive correlation means that as one variable increases, the other also increases, while a negative correlation (also called an anticorrelation) means that as one variable increases, the other decreases.

[0078] By "corresponding to" or "corresponding to" is meant an amino acid sequence that exhibits substantial sequence similarity or identity with a reference amino acid sequence. Generally, an amino acid sequence will exhibit at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even up to 100% sequence similarity or identity with at least a portion of a reference amino acid sequence.

[0079] As used herein, the term "cytotoxic agent" refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise interferes with cell function). Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 、Y 90 、Re 186 、Re188 、Sm 153 、Bi 212 、P 32 , Pb 212 and radioisotopes of Lu); chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Exemplary cytotoxic agents can be selected from antimicrotubule agents, platinum coordination compounds, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In some embodiments, the cytotoxic agent is a taxane. In representative examples of this type, the taxane is paclitaxel or docetaxel. In some embodiments, the cytotoxic agent is a platinum agent. In some embodiments, the cytotoxic agent is an antagonist of EGFR. In a representative example of this type, the antagonist of EGFR is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In some embodiments, the cytotoxic agent is a RAF inhibitor. In a non-limiting example of this type, the RAF inhibitor is a BRAF and / or CRAF inhibitor. In other non-limiting examples, the RAF inhibitor is vemurafenib. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

[0080] As used herein, the term "cytotoxic therapy" refers to a therapy that induces cell damage, including but not limited to radiation therapy, chemotherapy, photodynamic therapy, radiofrequency ablation, anti-angiogenic therapy, and combinations thereof. When cytotoxic therapeutics are applied to cells, they may induce DNA damage.

[0081] As used herein, "delaying disease progression" or "slowing the rate of disease progression" means postponing, hindering, slowing down, slowing down, stabilizing, and / or delaying the development of a disease (e.g., cancer). The duration of such delay can vary, depending on the history of the disease and / or the individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay can actually include prevention, i.e., the individual will not develop the disease. For example, progression to advanced cancer, such as the development of metastases, can be delayed.

[0082] The term "detecting" includes any means of detection, including direct and indirect detection.

[0083] As used herein, the term "drug" refers to any substance that has biological activity or detectable activity in vivo. The term drug is intended to include cytotoxic agents, cytostatic agents, anti-angiogenic agents, tumor reduction agents, chemotherapeutic agents, radiotherapeutic agents, targeted anticancer agents, biological response modifiers, cancer vaccines, cytokines, hormone therapy, anti-metastatic agents, and immunotherapeutic agents.

[0084] The term "drug resistance" refers to a condition where a disease does not respond to treatment with one or more drugs. Drug resistance can be intrinsic (or primary resistance), meaning that the disease has never responded to the drug or drugs, or it can be acquired, meaning that the disease no longer responds to one or more drugs to which the disease previously responded (secondary resistance). In certain embodiments, drug resistance is intrinsic. In certain embodiments, drug resistance is acquired.

[0085] "Effective amount" is at least the minimum amount required to achieve measurable improvement or prevention of a particular condition. The effective amount herein can vary according to factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount also refers to an amount when any toxicity or harmful effects of the treatment are offset by the beneficial effects of the treatment. For preventive applications, the results that are beneficial or desired include, for example, biochemical, histological, and / or behavioral symptoms of the disease, its complications, and the intermediate pathological phenotypes presented during the course of the disease, eliminating or reducing its risk, alleviating its severity, or delaying its onset. For therapeutic applications, the results that are beneficial or desired include clinical outcomes, such as reducing one or more symptoms caused by the disease, improving the quality of life of people with the disease, reducing the dosage of other drugs required for treating the disease, enhancing the effect of another drug (such as by targeted therapy), delaying the progression of the disease, and / or prolonging life. In the case of cancer or a tumor, an effective amount of a drug may have the following effects: reducing the number of cancer cells; reducing the size of the tumor; inhibiting (i.e., slowing down or desirably stopping to some extent) the infiltration of cancer cells into surrounding organs; inhibiting (i.e., slowing down and desirably stopping to some extent) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the cancer or tumor. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition refers to an amount sufficient to achieve, directly or indirectly, a preventive or therapeutic treatment. As understood in a clinical setting, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result may or has been achieved when used in combination with one or more other agents. The effective amount of treatment can be measured by evaluating various endpoints commonly used in cancer treatment, including but not limited to: prolonging survival (including overall survival (OS) and progression-free survival (PFS)); causing an objective response (including complete response (CR) or partial response (PR)); tumor regression, reduction in tumor weight or size, prolonged time before disease progression, prolonged survival duration, prolonged PFS, improved OS rate, prolonged duration of response, and improved quality of life and / or improved signs or symptoms of cancer. As used herein, the term "progressive disease" (PD) refers to an increase of at least 20% in the sum of the diameters of the target lesions, with the smallest sum of diameters in the study as a reference (including the baseline sum if the baseline sum is the smallest in the study). In addition to a relative increase of 20%, an absolute increase of at least 5 mm in the sum must also be demonstrated. The appearance of one or more new lesions is also considered to be progress.As used herein, the term "partial response" (PR) refers to a reduction of at least 30% in the sum of the diameters of target lesions, relative to the baseline sum of the diameters. As used herein, the term "complete response" (CR) refers to the disappearance of all non-nodal target lesions and the reduction of the short axis of any target lymph node to <10 mm. As used herein, the term "stable disease" (SD) refers to a patient with neither sufficient reduction to qualify as a PR nor sufficient increase to meet the criteria for a PD, relative to the smallest sum of the diameters on study.

[0086] The term "epitope" refers to a part of a molecule that can be recognized and bound by an antibody at one or more antigen-binding portions of an antibody. An epitope is often composed of surface molecular clusters such as amino acids or sugar side chains and has specific three-dimensional structural characteristics and specific charge characteristics. In some embodiments, the epitope can be a protein epitope. A protein epitope can be linear or conformational. In a linear epitope, all interaction points between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises a non-continuous polypeptide (or amino acid) in an antigenic protein that is bound by an epitope-specific antibody. Once the desired epitope on the antigen is determined, it is possible to produce antibodies against the epitope, for example, using the technology described in this specification. Alternatively, during the exploratory process, the generation and characterization of antibodies can elucidate information about the desired epitope. Based on this information, it is then possible to competitively screen antibodies that bind to the same epitope. A means to achieve this goal is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with each other for binding to the target antigen (e.g., EOMES-641K-Ac, EOMES-641K-Me, EOMES-373K-Me, etc.), e.g., the antibodies compete for binding to the antigen.

[0087] The term "expression" with respect to a gene sequence refers to the transcription of the gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.), and, where appropriate, the translation of the resulting mRNA transcript into a protein. Thus, it is clear from the context that the expression of a coding sequence results from both the transcription and translation of the coding sequence. In contrast, the expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0088] As used herein, the term "increase" or "increase" with respect to the level of a biomarker or biomarker complex refers to a statistically significant and measurable increase in the level of the biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or a control level. The increase is preferably an increase of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%.

[0089] As used herein, the term "higher" with respect to a measured value of a biomarker or biomarker complex refers to a statistically significant and measurable difference in the level of a measured value of one biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or to a control level, wherein the measured value of the biomarker or biomarker complex is greater than the level of the other biomarker or biomarker complex or the control level. The difference is preferably at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%.

[0090] As used herein, the term "reduction" or "reduction" with respect to the level of a biomarker or biomarker complex refers to a statistically significant and measurable decrease in the level of the biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or a control level. The decrease is preferably at least about a 10% decrease, or at least about a 20% decrease, or at least about a 30% decrease, or at least about a 40% decrease, or at least about a 50% decrease.

[0091] As used herein, the term "lower" with respect to a measured value of a biomarker or biomarker complex refers to a statistically significant and measurable difference in the level of a measured value of one biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or to a control level, wherein the measured value of the biomarker or biomarker complex is less than the level of the other biomarker or biomarker complex or the control level. The difference is preferably at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%.

[0092] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a sample. "Expression" generally refers to the process by which information (e.g., genetically encoded and / or epigenetic) is converted into a structure that exists and operates in a cell. Thus, as used herein, "expression" can refer to transcription into polynucleotides, translation into polypeptides, or even polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides). Fragments of transcribed polynucleotides, translated polypeptides, or polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered to be expressed, whether they are derived from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as by proteolysis. "Expressed genes" include genes that are transcribed into polynucleotides in the form of mRNA and then translated into polypeptides, as well as genes that are transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA). Thus, "increased expression," "increased expression levels," or "increased levels" refer to an increase in expression or an increase in the level of a biomarker in a cell or individual relative to a control, such as a cell or cells that respond or do not respond to a therapy, or an individual or individuals that respond or do not respond to a therapy, or an internal control (e.g., a housekeeping biomarker). "Decreased expression," "decreased expression levels," or "decreased levels" refer to a decrease in expression or a decrease in the level of a biomarker in an individual relative to a control, such as a cell or cells that respond or do not respond to a therapy, or an individual or individuals that respond or do not respond to a therapy, or an internal control (e.g., a housekeeping biomarker). In some embodiments, decreased expression is a small amount of expression or no expression. In specific embodiments, increased levels of EOMES-641K-Ac refer to levels associated with most nuclear localization of EOMES, or localization in the nucleus is higher than localization in the cytoplasm. Elevated levels of EOMES-641K-Ac can also be associated with resistance to therapy. In other embodiments, elevated EOMES-641K-Me levels refer to levels associated with a predominantly cytoplasmic localization of EOMES, or a localization in the cytoplasm and / or cell membrane that is higher than in the nucleus. Elevated EOMES-641K-Me levels can also be correlated with responsiveness to therapy.

[0093] The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and / or polypeptides) whose presence is generally similar in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene." "Housekeeping gene" herein refers to a gene or a group of genes that encode proteins whose activity is essential for maintaining cellular function and whose presence is generally similar in all cell types.

[0094] As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits cell growth in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces the proliferation of cells expressing the antigen to which the antibody binds. In another embodiment, the growth inhibitory agent can be an agent that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at stages other than the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include Vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest the G1 phase can also spread to S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and cytarabine. More information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, by Murakami et al., entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders, Philadelphia, 1995), e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs extracted from the yew tree. Docetaxel ( Rhone-Poulenc Rorer) is derived from the European yew and is a taxol ( Paclitaxel and docetaxel are semisynthetic analogs of tubulin (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, thereby inhibiting cell mitosis.

[0095] The term "immune checkpoint molecules" includes receptors and ligands that function as immune checkpoints. Immune checkpoints represent immune escape mechanisms that prevent the immune system from attacking itself. Immune checkpoint receptors are present on T cells and interact with immune checkpoint ligands expressed on antigen-presenting cells (including cancer cells). T cells recognize antigens presented on MHC molecules and are activated to produce an immune response, and the interaction between immune checkpoint receptors and ligands that occurs simultaneously with the above controls the activation of T cells. Immune checkpoint receptors include co-stimulatory receptors and inhibitory receptors, and the activation of T cells and the immune response are controlled by the balance between the two receptors. Illustrative immune checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed in all NK, gamma delta, and memory CD8 + (αβ) expressed on T cells), CD160 (also known as BY55), and CGEN-15049.

[0096] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to any agent, molecule, compound, chemical, protein, polypeptide, macromolecule, etc. that reduces, inhibits, interferes with, or modulates, in whole or in part, one or more immune checkpoint molecules. Such inhibitors may include small molecule inhibitors, or may include antigen binding molecules that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrative immune checkpoint inhibitors include anti-immune checkpoint molecule antagonist antibodies, such as, but not limited to, durvalumab (anti-PD-L1 antibody; MEDI4736), pembrolizumab (anti-PD-1 monoclonal antibody), nivolumab (anti-PD-1 antibody), pidilizumab (CT-011; humanized anti-PD-1 monoclonal antibody), AMP224 (recombinant B7-DC-Fc fusion protein), BMS-936559 (anti-PD-L1 antibody), atezolizumab (MPLDL3280A; human Fc-optimized anti-PD-L1 monoclonal antibody), avelumab (MSB0010718C; human anti-PD-L1 antibody), ipilimumab (anti-CTLA-4 checkpoint inhibitor), tremelimumab (CTLA-4 blocking antibody), and anti-OX40.

[0097] The term "immune effector cell" in the context of the present invention refers to a cell that plays an effector function during an immune response. For example, such cells secrete cytokines and / or chemokines, kill microorganisms, secrete antibodies, identify infected or cancerous cells, and optionally remove these cells. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, neutrophils, macrophages and dendritic cells.

[0098] The term "immune response" refers to any detectable response of the host mammal's immune system to a specific substance (e.g., an antigen or immunogen), such as an innate immune response (e.g., activation of the Toll receptor signaling cascade), a cell-mediated immune response (e.g., T cells of the immune system, such as antigen-specific T cells and non-specific cell-mediated responses), and a humoral immune response (e.g., a B cell-mediated response, such as the production and secretion of antibodies into plasma, lymph and / or tissue fluids).

[0099] Regarding T cells, especially CD8 + The term "immune function" or "function" of a T cell refers to the ability of a T cell to proliferate, become activated and / or lyse cells. The immune function of a T cell can be assessed using well-known functional assays that measure any one or more of proliferation, activation or lysis. In a specific example, the anti-tumor activity of a T cell is used to assess its immune function. Alternatively, the expression and / or secretion of various effector proteins in a T cell can be assessed, such as IFN-γ, TNF-α, IL-2, Ki67 or CD107a. For example, IFN-γ, IL-2 and TNF can be used as markers for CD8 + CD107a can be used as a biomarker for T cell activation; CD107a can be used as a biomarker for degranulation; and Ki67 can be used as a biomarker for T cell proliferation. + T cells or CD8 + A T cell population is a T cell that is capable of proliferating, being activated, and / or lytic at the levels expected for T cells from a healthy subject, as assessed using the assays and / or effector proteins / biomarkers described above and herein. In contrast, a dysfunctional T cell or T cell population, such as a dysfunctional CD8 + T cells or CD8 +A population of T cells whose ability to proliferate, become activated and / or become cytolytic is reduced or decreased, e.g., whose proliferation, activation and / or cytolysis is reduced or decreased at a level as assessed using the assays and / or effector proteins / biomarkers described above and herein (e.g., reduced or decreased by about or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to a functional T cell or T cell population).

[0100] The term "immunotherapy" refers to any of the following therapies in which one or more components of the immune system of a human or animal are deliberately modulated to obtain, directly or indirectly, some therapeutic benefit, including systemic and / or local effects, as well as preventive and / or therapeutic effects. Immunotherapy can include administering one or more immunotherapeutic agents, alone or in any combination, to a human or animal subject by any route (e.g., oral, intravenous, transdermal, by injection, by inhalation, etc.), whether systemic, local, or both. Immunotherapy can involve stimulating, increasing, decreasing, pausing, preventing, blocking, or otherwise regulating the production of cytokines, and / or activating or deactivating cytokines or immune cells, and / or regulating the level of immune cells, and / or delivering one or more therapeutic or diagnostic substances to a specific site or specific type of cell or tissue in the body, and / or destroying specific cells or tissues. Immunotherapy can be used to achieve local effects, systemic effects, or a combination of the two.

[0101] The term "immunotherapeutic agent" as used herein refers to any agent, compound or biological product that indirectly or directly restores, enhances, stimulates or increases the body's immune response to cancer cells and / or reduces the side effects of other anticancer therapies. Therefore, immunotherapy is a therapy that directly or indirectly stimulates or enhances the immune system's response to cancer cells and / or alleviates the side effects that may be caused by other anticancer agents. Immunotherapy is also referred to as immunotherapy, biological therapy, biological response modifier therapy and biotherapy in the art. Examples of conventional immunotherapeutics known in the art include but are not limited to cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, immunotherapy can consist of administering a certain amount of immune cells (T cells, NK cells, dendritic cells, B cells, etc.) to the subject. Immunotherapeutics can be nonspecific, i.e., broadly strengthen the immune system so that the human body can more effectively fight the growth and / or spread of cancer cells, or they can be specific, i.e., target the cancer cells themselves. Immunotherapy regimens can be combined with nonspecific and specific immunotherapeutics. Nonspecific immunotherapeutics are substances that stimulate or indirectly improve the immune system. Nonspecific immunotherapeutics have been used alone as the primary therapy for treating cancer, and as a supplement to the primary therapy. In this case, nonspecific immunotherapeutics act as adjuvants to enhance the effectiveness of other therapies (e.g., cancer vaccines). Nonspecific immunotherapeutics can also play a role in the latter case to reduce the side effects of other therapies, such as bone marrow suppression induced by certain chemotherapeutics. Nonspecific immunotherapeutics can act on key immune system cells and cause secondary responses, such as increasing the production of cytokines and immunoglobulins. Alternatively, the agent itself can contain cytokines. Nonspecific immunotherapeutics are generally divided into cytokines or non-cytokine adjuvants. Many cytokines have been used in cancer treatment and can be used as general nonspecific immunotherapies intended to strengthen the immune system, or as adjuvants provided together with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony stimulating factors. The interferons (IFNs) encompassed by the present invention include common types of IFNs, i.e., IFN-alpha (IFN-α), IFN-beta (IFN-β), and IFN-gamma (IFN-γ). IFN can act directly on cancer cells, for example, by slowing their growth, promoting them to develop into cells with more normal behavior and / or increasing their antigen production, thereby making it easier for the immune system to recognize and destroy cancer cells. IFN can also act indirectly on cancer cells, for example, by slowing down angiogenesis, strengthening the immune system and / or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-α is available on the market, such as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).Interleukins encompassed by the present invention include IL-2, IL-4, IL-11, and IL- 12. Examples of commercially available recombinant interleukins include. (IL-2; Chiron Corporation) and (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim), and erythropoietin (epoetin alfa, darbopoietin). In subjects receiving conventional chemotherapy, treatment with one or more growth factors helps stimulate the production of new blood cells. Accordingly, treatment with CSFs can help reduce chemotherapy-related side effects and allow the use of higher doses of chemotherapeutic agents. A variety of recombinant colony stimulating factors are commercially available, such as (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Aranesp (erythropoietin). In addition to having specific or nonspecific targets, immunotherapeutics can be active, i.e., stimulate the body's own immune response, including humoral and cellular immune responses, or they can be passive, i.e., involve immune system components, such as antibodies, effector immune cells, antigen-presenting cells, etc., that are produced outside the body. In specific embodiments, passive immunotherapy involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of cancer cells or immune cells, or for a particular cellular growth factor. Monoclonal antibodies can be used in a variety of ways to treat cancer, for example, to enhance a subject's immune response to a particular type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors (e.g., growth factors involved in angiogenesis), or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents (e.g., chemotherapeutic agents, radioactive particles, or toxins). Monoclonal antibodies currently used as cancer immunotherapeutics include, but are not limited to, alemtuzumab. Bevacizumab Cetuximab Panitumumab Pertuzumab ( 2C4), trastuzumab Tositumomab Abciximab Adalimumab Apolizumab, aselizumab, tocilizumab, bapineuzumab, basiliximab Bavituximab, belimumab briankinumab, canakinumab Cilizumab, Certolizumab cidfusituzumab, cidtuzumab, cixutumumab, clazakizumab, crenezumab, daclizumab Dalotuzumab, denosumab ), Eculizumab Efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, golimumab Ipilimumab, imgatuzumab, infliximab labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab, lulizumab pegol, lumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, motovizumab, muronomab, natalizumab Nexituzumab Nimotuzumab Nolovizumab, numavizumab, olokizumab, omalizumab Onartuzumab (also known as MetMAb), palivizumab Pascolizumab, pecfusituzumab, pectuzumab, pembrolizumab Pexelizumab, priliximab, ralvizumab, ranibizumab, ), reslivizumab, reslizumab, resyvizumab, robatumumab, rontalizumab, rovelizumab, ruplizmnab, sarilumab, secukinumab, seribantumab, sifalimumab, sibrotuzumab, siruximab Siplizumab, sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab toralizumab, tucusituzumab, umavizmab, urtoxazumab, and ustekinumab Vedolizumab visilizumab, zanolimumab, and zalutumumab.

[0102] As used herein, "instructional materials" include publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. For example, the instructional materials of the kit of the present invention can be attached to a container containing the therapeutic or diagnostic agent / agent of the present invention, or can be shipped together with a container containing the therapeutic or diagnostic agent / agent of the present invention.

[0103] As used herein, the term "label" refers to a detectable compound or composition. A label is typically conjugated or fused directly or indirectly to a reagent (e.g., a polynucleotide probe or antibody) and facilitates detection of the conjugated or fused reagent. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze a chemical change in a substrate compound or composition to produce a detectable product.

[0104] As used herein, the term "localize" and its grammatical equivalents mean accumulation or confinement to a specific or limited space or region, such as a specific cell, tissue, organelle, or intracellular region, such as a cellular compartment (e.g., nucleus, cytoplasm, nuclear membrane, cell membrane, etc.).

[0105] The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acid obtained from a single source (eg, an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.

[0106] The terms "patient," "subject," "host," or "individual" are used interchangeably herein to refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom treatment or prevention is desired. Suitable vertebrates within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys, and apes, and including species of monkeys such as from the genus Macaca (e.g., cynomolgus monkeys, such as Macaca fascicularis, and / or rhesus macaques (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri), and tamarins (species from the genus Saguinus), and species of apes such as chimpanzees (Pan The subject may be a mammal (e.g., a troglodytes), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. Preferred subjects are humans suffering from cancer.

[0107] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in a form that permits the biological activity of the active ingredient(s) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition or formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those substances that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0108] The term "predictive," and its grammatical forms, generally refers to a biomarker or biomarker signature that provides a means of directly or indirectly identifying the likelihood that a patient will respond to a therapy or achieve a clinical outcome in response to a therapy.

[0109] The term "prognosis" and its grammatical forms generally refer to an agent / reagent or method that provides information about the possible progression or severity of a disease or condition in an individual. In some embodiments, prognosis also refers to the ability to show a positive or negative response to a therapy or other treatment regimen for a subject's disease or condition. In some embodiments, prognosis refers to the ability to predict the presence or alleviation of symptoms associated with a disease / condition. Prognostic agents / reagents or methods can include dividing a subject or a sample obtained from a subject into one of a variety of categories, wherein these categories are associated with different probabilities that the subject will experience a particular outcome. For example, categories can be low risk and high risk, wherein the subject in the low-risk category has a lower likelihood of experiencing an adverse outcome than the subject in the high-risk category (e.g., within a given time period, such as 5 or 10 years). Adverse outcomes may be, for example, progression of disease, recurrence of disease, or death attributable to the disease.

[0110] By "radiation therapy" is meant the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to completely destroy the cells. It will be appreciated that there are many known ways in the art to determine the dosage and duration of treatment. Typical treatment is given as a one-time administration, with typical dosages ranging from 10 to 200 units (Gy) per day.

[0111] As used herein, a cancer patient (or subject with cancer) who is being treated with a therapy is considered to be "responsive," "responsive," "positively responsive," or "responding" to a therapy if the subject shows evidence of an anti-cancer effect, including a clinically significant benefit, such as preventing or reducing the severity of cancer symptoms, or slowing the progression of the cancer, according to a set of objective criteria recognized in the art, or a reasonable modification thereof. It will be understood that the above terms can also be used in the context of cancer. A variety of different objective criteria for evaluating the effect of anti-cancer treatments on cancer are known in the art. The World Health Organization (WHO) criteria (Miller, AB et al., Cancer 1981; 47(1): 207-14) and its improved version, the Response Evaluation Criteria in Solid Tumors (RECIST) (Therasse P et al., J Natl Cancer Inst 2000; 92: 205-16) and its revised version (Eisenhauer EA, New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45(2): 228-47) are several sets of objective criteria based on imaging measurements of the size and number of tumor lesions and the detection of new lesions, such as from computed tomography (CT), magnetic resonance imaging (MRI) or conventional radiographs. The size of the selected lesions (called target lesions) is used to calculate the change in tumor burden between images at different time points. The calculated response is then classified as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). CR is the complete disappearance of the tumor (-100%), while PD is an increase of approximately 20%-25% or greater (depending on the specific criteria) and / or the appearance of new lesions. PR is a significant decrease in the size of tumor lesions (at least about 30%) (without the appearance of new lesions), but a complete response is not achieved. SD is between PR and PD. (See Tables 1 and 2 for details.) These criteria are widely used as primary endpoints for evaluating the efficacy of anticancer agents in Phase II clinical trials, for example as surrogate markers of overall survival. However, anatomical imaging alone using WHO, RECIST, and RECIST 1.1 criteria is designed to detect the early effects of cytotoxic agents and has certain limitations, particularly in evaluating the activity of new cancer therapies in patients with stable disease. The clinical response pattern of patients receiving immunotherapeutic anticancer agents or molecularly targeted anticancer agents may extend beyond the range of cytotoxic agents and may manifest after the initial increase in tumor burden or the appearance of new lesions. For example, a meaningful tumor response to immune checkpoint inhibitors may occur after a delay, in some cases after PD as defined by WHO or RECIST.Defined a standard called immune-related response criteria (irRC), attempting to capture other favorable response patterns observed in immunotherapy (Wolchok, JD et al. (2009) Guidelines for the evaluation of immune therapy activity in solid tumors:immune-related response criteria.Clin.Care Res.15,7412-7420.). Four patterns associated with favorable survival were identified, namely, a reduction in baseline lesions and no new lesions; persistent stable disease; an initial increase in total tumor load, but ultimately a response; and a reduction in total tumor load during or after the appearance of new lesions (one or more), the latter two of which are different from the response patterns considered favorable according to WHO or RECIST criteria. irRC includes criteria for complete response (irCR), partial response (irPR), stable disease (irSD) and progressive disease (irPD). Among other things, irRC incorporates measurable new lesions into the "total tumor load" and compares this variable with baseline measurements, rather than assuming that new lesions necessarily represent progressive disease. In summary, according to immune-related response criteria, irCR refers to the complete disappearance of all lesions, whether measurable or not, with no new lesions; irPR is a reduction in tumor burden of ≥50% relative to baseline; irSD refers to disease that does not meet the criteria for irCR or irPR in the absence of progressive disease (irPD); irPD is an increase in tumor burden of ≥25% relative to the nadir (the minimum tumor burden recorded) (Wolchok, see above). irCR, irPR, and irPD require confirmation by repeated, serial assessments at least 4 weeks from the date of first recording. irCR, irPR, and irSD include all patients with CR, PR, or SD who meet WHO criteria, as well as patients who transition from WHO PD to these irRC categories. However, some patients who would be classified as having PD according to WHO or RECIST criteria are instead classified as PR or SD according to irRC, thereby determining that they may have favorable survival. irRC is applicable to immune checkpoint inhibitors and other immunotherapeutic agents. Those skilled in the art will appreciate that other response criteria are known in the art that take into account various factors, such as changes in the extent of tumor arterial enhancement and / or tumor density as indicators of viable tumor tissue, where decreased arterial enhancement and decreased tumor density are indicators of decreased viable tumor tissue (e.g., due to tumor necrosis). For example, the modified RECIST criteria (mRECIST) take into account changes in the extent of tumor arterial enhancement (Lencioni R and Llovet JM. Semin Liver Dis 30:52-60, 2010).The Choi criteria and the modified Choi criteria take into account the reduction of tumor density on CT. Choi H et al., J Clin Oncol 25:1753-1759, 2007; Nathan PD et al., Cancer Biol Ther 9:15-19, 2010; Smith AD et al., Am J Roentgenol 194:157-165, 2010. Such criteria may be particularly useful for certain cancer types and / or certain categories of therapeutic agents. For example, although treatment is effective, in tumors such as lymphoma, sarcoma, hepatoma, mesothelioma and gastrointestinal stromal tumors, the change in tumor size may be minimal. CT tumor density, contrast enhancement or MRI features appear to be more valuable than size. In certain embodiments, functional imaging, such as positron emission tomography (PET), can be used. For example, PET response criteria (PERCIST) can be used for solid tumors, where treatment response is assessed by metabolic changes assessed using (18)F-FDG PET imaging, with a decrease in tracer uptake as an indicator (Wahl RL et al., J Nucl Med 2009;50, Suppl 1:122S-50S). It should also be understood that response criteria developed for various specific cancer types, such as melanoma, breast cancer, and lung cancer, are known in the art. In contrast, a cancer patient who has been treated with a therapy is considered to be "unresponsive," "lacking a response," having a "negative response," or "non-responsive" to a therapy if the therapy does not provide a clinically significant benefit, such as preventing or reducing the severity of symptoms, or increases the rate of progression of the cancer.

[0112] For the purposes of this disclosure, for cancer patients receiving immunotherapy (e.g., immune checkpoint inhibitors) as monotherapy or in combination with one or more other active agents (e.g., complement inhibitors, other anticancer agents, or both), at least according to immune-related response criteria, if the patient has a complete response, partial response, or stable disease, then the patient is considered to be "responsive", "responsive" or "responsive" to the treatment. (Cancer patients may also respond according to RECIST, RECIST 1.1, WHO, and / or other criteria such as those mentioned above.) Similarly, the cancer in this case is referred to as "responsive", "responsive" or "sensitive" to the treatment. According to immune-related response criteria, if the patient has progressive disease, the cancer patient is considered to be "non-responsive", "non-responsive" or "non-responsive" to the treatment. (Cancer patients may also be non-responsive according to RECIST, RECIST 1.1, WHO, and / or other criteria such as those mentioned above.) Similarly, the cancer in this case is referred to as "non-responsive", "non-responsive", "non-sensitive" or "resistant" to the treatment. (Cancer is also considered to be resistant to treatment if, in the presence of treatment, the patient initially responds but then shows progression of the disease.) Thus, for example, for methods and products described herein related to responses to cancer treatments (e.g., methods for predicting the likelihood of a response, methods for classifying patients based on predicted responses, methods for increasing the likelihood of a response), unless otherwise specified, responses are defined as irCR, irPR, or irSD, and lack of response is defined as irPD. In certain embodiments, any useful response criteria may be specified. Response criteria may have been shown to be associated with benefits, such as an increase in overall survival or other clinically significant benefits. It should be understood that existing response criteria may be improved or revised in the future, for example, including other favorable modes of clinical activity applicable to immune checkpoint inhibitors (e.g., associated with an extension of overall survival) or criteria that are useful in other respects. In certain embodiments, any such response criteria may be specified for use in the methods described herein.

[0113] As used herein, the term "sample" includes any biological specimen that can be extracted, untreated, processed, diluted or concentrated from a subject. Sample includes within its scope a collection of similar fluids, cells or tissues (e.g., surgically removed tumor tissue, biopsy, including fine needle aspiration) separated from a subject, and fluids, cells or tissues present in a subject. In some embodiments, the sample is a biological fluid. Typically, a biological fluid is a liquid at physiological temperature and can include a naturally occurring fluid that is present in a subject or a biosource, taken from a subject or a biosource, expressed from a subject or a biosource, or otherwise extracted from a subject or a biosource. Some biological fluids derive from specific tissues, organs or local regions, while other biological fluids may be more comprehensively or systematically located in a subject or a biosource. Examples of biological fluids include blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as ascites associated with non-solid tumors, fluids from the pleura, pericardium, peritoneum, abdomen and other body cavities, fluids collected from bronchial lavage, etc. Biological fluids can also include liquid solutions that are in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media, lavage fluids, etc. As used herein, the term "sample" includes material removed from a subject or material present in a subject.

[0114] As used herein, "reference sample," "reference cell," "reference tissue," "reference level," "control sample," "control cell," "control tissue," or "control level" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased body part (e.g., tissue or cell) of the same subject or individual, but at a different time point, e.g., before and after treatment. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy individual who is not the subject or individual being evaluated. In a specific example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is or comprises functional T cells, dysfunctional T cells (e.g., exhausted T cells), T cells from a subject who is responsive or sensitive to therapy, or T cells from a subject who is unresponsive or resistant to therapy. In a specific embodiment, the T cells are CD8 + T cells.

[0115] Various methodologies of the present invention include the following steps, which involve comparing values, levels, features, characteristics, attributes, etc. with an "appropriate control", which is interchangeably referred to herein as a "suitable control", "control sample" or "reference". An "appropriate control", "suitable control", "control sample" or "reference" is any control or standard for comparison purposes familiar to those of ordinary skill in the art. In some embodiments, an "appropriate control" or "suitable control" is a value, level, feature, characteristic, attribute, etc. determined in a cell, tissue or patient (e.g., a control cell, a cell population, a tissue or a patient) that exhibits, for example, a specific biomarker spectrum. An "appropriate control" can be a level / ratio pattern of one or more biomarkers of the present invention that is associated with a specific biomarker spectrum, and a cell sample can be compared with the biomarker spectrum. A cell sample can also be compared with a negative control. Such reference levels can also be customized according to the specific technology used to measure the levels of biomarkers in a biological sample (e.g., LC-MS, GC-MS, ELISA, PCR, etc.), wherein the levels of the biomarkers may vary based on the specific technology used. Suitable controls can include, for example, functional T cells, dysfunctional T cells (e.g., exhausted T cells), T cells from subjects who are responsive or sensitive to cancer therapy, and T cells from subjects who are unresponsive or resistant to cancer therapy.

[0116] As used herein, the terms "stratification" and "classification" are used interchangeably herein and refer to the classification of subjects into different levels or categories based on the characteristics of a particular physiological or pathophysiological state or condition. For example, a subject population is stratified based on whether the subject is likely to respond to therapy (e.g., chemotherapy or immunotherapy), which involves allocating subjects based on the level of therapy response biomarkers (including EOMES-641K-Ac, EOMES-641K-Me, and EOMES-373K-Me) in T cells, optionally in combination with one or more other biomarkers (e.g., IFN-γ, TNF-α, IL-2, Ki67, PD-1, or CD107a).

[0117] As used herein, the term "treatment" refers to a clinical intervention intended to change the natural course of an individual or cell being treated in a clinical pathological process. The desired effect of treatment includes reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing the proliferation of cancerous cells (or destroying cancer cells), reducing pathogen infection, alleviating the symptoms caused by the disease, improving the quality of life of the people with the disease, reducing the dosage of other drugs required for treating the disease and / or prolonging the survival period of the individual, the individual is successfully "treated". Phrases such as "treating with therapy", "treating with therapy", "treating with a medicament", "treating with a medicament" refer to administering an effective amount of therapy or medicament to the patient, including cancer therapy or medicament (for example, cytotoxic agent or immunotherapeutic agent), or administering an effective amount of two or more therapies or medicaments to the patient together, including cancer therapy or medicament (for example, two or more medicaments selected from cytotoxic agent and immunotherapeutic agent).

[0118] As used herein, "treatment outcome" refers to a prediction of a cancer patient's response to a selected therapy or treatment, including the likelihood that the patient will experience a positive or negative outcome after receiving a particular treatment. As used herein, "indicative of a positive treatment outcome" or similar phrases refers to an increased likelihood that the patient will experience a beneficial outcome from the selected treatment (e.g., complete or partial response, complete or partial remission, reduction in tumor size, stable disease, etc.). Conversely, "indicative of a negative treatment outcome" or similar phrases is intended to indicate an increased likelihood that the patient will not benefit from the selected treatment in terms of progression of the underlying cancer (e.g., disease progression, disease recurrence, increase in tumor size, etc.).

[0119] As used herein, "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorders," "proliferative disorders," "hyperproliferative disorders," and "tumor" as used herein are not mutually exclusive.

[0120] As used herein, underlining or italics of a gene name shall indicate the gene, not its protein product, and the name of the gene in the absence of any underlining or italics shall indicate its protein product. For example, "EOMES" shall mean the EOMES gene, and "EOMES" shall mean the protein product(s) produced by transcription and translation and / or alternative splicing of the EOMES gene.

[0121] Unless specifically stated otherwise, each embodiment described herein applies mutatis mutandis to each and every embodiment.

[0122] 2. Methods of detection, diagnosis, and prognosis

[0123] EOMES is a transcription factor associated with T cell exhaustion and dysfunction. The present invention discloses that different post-translational modifications of lysine 641 (contained in the EOMES NLS) or lysine 373 (contained in the EOMES DNA binding domain) in the EOMES polypeptide sequence affect EOMES localization to the nucleus or cytoplasm and may also affect protein:protein or protein:DNA interactions. Furthermore, EOMES polypeptides with different post-translational modifications of these lysines are associated with functional or dysfunctional T cells and can be used to predict responsiveness to cancer therapies.

[0124] A representative EOMES polypeptide comprises the following amino acid sequence:

[0125]

[0126] YTTP [SEQ ID NO: 1], wherein lysine at position 373 (i.e., 373K) and lysine at position 641 (i.e., 641K)

[0127] Highlighted in bold font.

[0128] The present inventors have found that acetylation of lysine 641 (ie, EOMES-641K-Ac) localizes EOMES primarily to T cells (e.g., CD8 + In addition, EOMES-641K-Ac expression is associated with dysfunctional T cells, with exhausted, aging T cell signatures (e.g., low or reduced expression of Ki67, TNF-α, and / or IFN-γ). Consistent with this finding, EOMES-641K-Ac expression is associated with resistance or non-responsiveness to cancer therapy.

[0129] The present inventors also found that methylation of lysine 641 in EOMES (i.e., EOMES-641K-Me) or methylation of lysine 373 in EOMES (i.e., EOMES-373K-Me), including dimethylation (Me2), is associated with functional T cell phenotypes and responsiveness to cancer therapy. These methylated forms of EOMES tend to be more localized in the cytoplasm than in the nucleus, although some nuclear expression is also observed.

[0130] Therefore, according to the present invention, EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me can be used as biomarkers for assessing T cell function, predicting a subject's likelihood of response to cancer therapy (e.g., chemotherapy and / or immunotherapy), including the likelihood of resistance or sensitivity to therapy, stratifying cancer patients into likely responders or non-responders to therapy, managing cancer patients' treatment with therapy, and predicting treatment outcomes in cancer patients receiving therapy.

[0131] T cells used to implement the present invention can be obtained from any suitable patient sample containing T cells, illustrative examples of which include liquid biopsies, tumor biopsies, primary cell cultures or cell lines derived from T cells, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. In some embodiments, the sample is obtained before treatment with therapy. In other embodiments, the sample is obtained after treatment with therapy. In some embodiments, the sample comprises a tissue sample, which can be formalin-fixed and paraffin-embedded, archived, fresh or frozen. In some embodiments, the sample is whole blood. In specific embodiments, the T cells are CD8 + T cells.

[0132] The presence and / or level / amount of a biomarker (e.g., any one or more of EOMES-641K-Ac, EOMES-641K-Me, and EOMES-373K-Me, and optionally one or more other biomarkers, such as biomarkers of T cell function, such as IFN-γ, TNF-α, IL-2, Ki67, PD-1, or CD107a) can be qualitatively and / or quantitatively determined according to any appropriate criteria known in the art, including but not limited to proteins and protein fragments. In certain embodiments, the presence and / or expression level / amount of a biomarker in a first sample is increased or elevated compared to the presence / absence and / or expression level / amount in a second sample (e.g., before treatment with a therapy). In certain embodiments, the presence / absence and / or level / amount of a biomarker in a first sample is decreased or reduced compared to the presence and / or level / amount in a second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Other disclosures for determining the presence / absence and / or level / amount of a gene are described herein.

[0133] In some embodiments of any of the methods, an increase in level refers to an overall increase in the level of a biomarker (e.g., protein or nucleic acid) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to a reference sample, reference cell, reference tissue, control sample, control cell or control tissue by standard methods known in the art, such as those described herein. In certain embodiments, an increase in level refers to an increase in the level / amount of a biomarker in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold or 100-fold the level / amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell or control tissue. In some embodiments, elevated levels refer to an overall increase of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, a reference cell, a reference tissue, a control sample, a control cell, a control tissue, or an internal control (e.g., a housekeeping gene).

[0134] In some embodiments of any of the methods, a reduction in level refers to an overall reduction in the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene or mRNA)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue by standard methods known in the art, such as those described herein. In certain embodiments, a reduction in level refers to a decrease in the level / amount of a biomarker in a sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold the level / amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0135] The presence and / or level / amount of various biomarkers in a sample can be analyzed by numerous methodologies, many of which are known in the art and understood by those of skill in the art, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzymatic activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") (including real-time quantitative PCR ("qRT-PCR") and other amplification type detection methods, such as branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as any of a wide variety of assays that can be performed by protein, gene and / or tissue array analysis. Typical protocols for assessing the status of genes and gene products are found in, for example, Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis). Multiplex immunoassays, such as those provided by Rules Based Medicine or MesoScale Discovery ("MSD"), can also be used.

[0136] In some embodiments, the following methods can be used to determine the presence and / or level / amount of biomarkers, particularly for biomarkers that are not characterized by post-translational modification (e.g., IFN-γ, TNF-α, IL-2, Ki67, PD-1, or CD107a), the method comprising: (a) performing gene expression profiling, PCR (e.g., RT-PCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on the sample; and b) determining the presence and / or expression level / amount of the biomarker in the sample. In some embodiments, the microarray method comprises using a microarray chip having one or more nucleic acid molecules or one or more polypeptides (e.g., peptides or antibodies), which can hybridize under stringent conditions with nucleic acid molecules encoding the above-mentioned genes, and the polypeptide can bind to one or more proteins encoded by the above-mentioned genes. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

[0137] Methods for assessing mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled riboprobes specific for one or more genes, Northern blot hybridization, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using complementary primers specific for one or more genes and other amplification-type detection methods, such as branched DNA, SISBA, TMA, etc.).

[0138] mRNA analysis can be conveniently performed on samples from mammals using Northern, dot blot, or PCR analysis. In addition, such methods can include one or more steps that allow one to determine the level of a target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined.

[0139] Optional method comprises such scheme, wherein by microarray technology inspection or detection tissue or cell sample in mRNA, such as target mRNA.Use nucleic acid microarray, to the test and control mRNA sample from test and control tissue sample is reverse transcribed and labeled, to produce cDNA probe.Then, probe and the nucleic acid array hybridization that is fixed on solid support.This array is configured to make the order and position of each member of this array be known.For example, can arrange one group of selected gene on solid support, the expression of these genes and the increase or reduction of the clinical benefit of therapy are relevant.The probe of labeling hybridizes with specific array member, shows that the sample that probe is derived from has expressed this gene.

[0140] In a preferred embodiment, presence and / or level / amount are measured by observing protein levels. In certain embodiments, the method includes making a biological sample (e.g., a sample from a cancer patient) and a biomarker (e.g., EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me) specific antigen binding molecule for therapy response contact under conditions allowing binding biomarker (one or more), and detecting whether a complex is formed between the antigen binding molecule or molecule and the biomarker (one or more). Such methods can be in vitro methods or in vivo methods. In some embodiments, one or more anti-biomarker antigen binding molecules are used to select a subject meeting therapy (e.g., immunotherapy) conditions.

[0141] In certain embodiments, the presence and / or expression level / amount of biomarker proteins in a sample are examined using immunohistochemistry (IHC) or immunofluorescence microscopy (IF) protocols. In some embodiments, the level of a therapy response biomarker (e.g., EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me) in a sample from an individual is an elevated level, and in further embodiments, IHC or IF are used to determine. In one embodiment, the level of a biomarker is determined using a method comprising the following: (a) performing IHC or IF analysis of a sample (e.g., a sample from a cancer patient) using an antigen binding molecule; and b) determining the level of a biomarker in a sample. In some embodiments, the staining intensity of IHC or IF is determined relative to a reference. In some embodiments, a reference is a reference value. In some embodiments, a reference is a reference sample (e.g., a control cell line staining sample or a sample from a non-cancerous patient or a sample from a patient before treatment).

[0142] In a specific method, the sample can be contacted with an antigen binding molecule specific for the biomarker under conditions sufficient to form a molecule-biomarker complex, and then the complex is detected. The presence of biomarkers can be detected in a variety of ways, such as by microscopy (e.g., IF microscopy), Western blotting, and ELISA procedures, for measuring a wide variety of tissues and samples, including blood. A wide range of immunoassay techniques using this assay format are available from, for example, U.S. Patents 4,016,043, 4,424,279, and 4,018,653. These include single-site and dual-site or "sandwich" assays of non-competitive types, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0143] In certain embodiments, the sample is standardized for the difference in the amount of the biomarker measured and the variability of the sample quality used, as well as the variability between the assay runs. This standardization can be achieved by detecting and incorporating the expression of certain standardized biomarkers (including the expression products of well-known housekeeping genes). Alternatively, standardization can be based on the average or median signal of all assay genes or a large subset thereof (global standardization method). On a gene-by-gene basis, the standardized amount of the measured subject's tumor mRNA or protein is compared with the amount found in the reference set. The standardized expression level of each mRNA or protein of each tested tumor of each subject can be expressed as a percentage of the expression level measured in the reference set. The presence and / or expression level / amount measured in the specific subject sample to be analyzed will fall within a certain percentage point within this range, which can be determined by methods well known in the art.

[0144] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual that is obtained at one or more different time points than when the test sample was obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at an earlier time point than when the test sample was obtained. Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained before treatment and the test sample is subsequently obtained after treatment.

[0145] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not a subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals who are not a subject or individual and have a disease or condition, such as cancer.

[0146] In some embodiments, the sample is a clinical sample. In some embodiments, the sample is a liquid biopsy, such as blood. In other embodiments, the sample is a tissue sample, such as a tumor tissue sample (e.g., biopsy tissue) containing T cells. In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes and / or bone / bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (eg, glioblastoma, neuroblastoma, etc.).

[0147] In some embodiments, tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, tumor and / or cancer is a solid tumor or non-solid or soft tissue tumor. The example of soft tissue tumor includes leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T cell lymphoma or Hodgkin's disease). Solid tumors include any body tissue cancer except blood, bone marrow or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basaloid colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian cancer), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary organs (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumors. In some embodiments, cancer is non-small cell lung cancer (NSCLC). In some embodiments, cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, cancer is adenocarcinoma. In some embodiments, cancer is squamous cell carcinoma. In some embodiments, cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC) or hepatocellular carcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site derived from any of the above types of cancer.

[0148] In some embodiments, at least one therapy response biomarker is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. In some embodiments, at least one therapy response biomarker is detected using FACS analysis or immunofluorescence microscopy. In some embodiments, at least one therapy response biomarker is detected in a blood sample. In some embodiments, the CD8 +At least one therapy response biomarker is detected in T cells.Any appropriate method can be used to separate / enrich this cell population, including but not limited to cell sorting.In some embodiments, in the sample from the individual for which response is produced by treatment with therapy, the expression of EOMES-641K-Ac is reduced, and suitably the therapy is immunotherapy (for example, a therapy comprising anti-immune checkpoint molecule antibodies).In some embodiments, in the sample from the individual for which treatment with therapy is not responded or responds weakly, the expression of EOMES-641K-Ac is increased, and suitably the therapy is immunotherapy (for example, a therapy comprising anti-immune checkpoint molecule antibodies).In some embodiments, in the sample from the individual for which treatment with therapy is not responded or responds weakly, the expression of EOMES-641K-Me and / or EOMES-373K-Me is reduced, and suitably the therapy is immunotherapy (for example, a therapy comprising anti-immune checkpoint molecule antibodies). In some embodiments, the expression of EOMES-641K-Me and / or EOMES-373K-Me is elevated in a sample from an individual that responds to treatment with therapy, and the therapy is suitably an immunotherapy (e.g., a therapy comprising an anti-immune checkpoint molecule antibody). In a specific example, the ratio of biomarkers is assessed, such as the ratio of EOMES-641K-Ac to EOMES-641K-Me, or vice versa.

[0149] In some embodiments, the expression level of one or more biomarkers can be compared with a reference, which can include, for example, a sample comprising functional or active T cells, a sample comprising dysfunctional or exhausted T cells, a sample from a subject not suffering from cancer, or a sample from a subject suffering from cancer but not receiving treatment (e.g., cytotoxic therapy or immunotherapy). In some embodiments, a reference can include a reference value from multiple subjects or samples. For example, as a whole, a mean, average, or median value of the expression level of at least one therapy response biomarker can be generated from a healthy subject population, a subject population that responds to therapy, or a subject population that does not respond to therapy, or from multiple samples of T cells of known immune function. A group of samples obtained from cancers with common characteristics (e.g., the same cancer type and / or stage, or receiving conventional therapy) can be studied from a population, for example, using clinical outcome studies. This group can be used to derive a reference, such as a reference value, with which the subject's sample can be compared.

[0150] Certain aspects of the present disclosure relate to measuring the expression level of one or more biomarkers (e.g., gene expression products, including mRNA and protein) in a sample comprising T cells. In some embodiments, the sample can be a peripheral blood sample (e.g., from a patient with cancer). In some embodiments, the sample is a tumor sample. In some embodiments, the sample can be processed to separate or isolate one or more cell types (e.g., CD8 T cells ... + T cells). In some embodiments, the sample can be used without separating or isolating the cell types.

[0151] Tumor samples can be obtained from subjects by any method known in the art, including but not limited to biopsy, endoscopy or surgery. In some embodiments, tumor samples can be prepared by methods such as freezing, fixing (e.g., by using formalin or similar fixatives) and / or embedding in solid paraffin. In some embodiments, tumor samples can be sliced. In some embodiments, fresh tumor samples (i.e., samples not prepared by the above method) can be used. In some embodiments, peripheral blood samples can be prepared by incubating in a solution to maintain the integrity of mRNA and / or protein.

[0152] In some embodiments, the sample can be a peripheral blood sample. The peripheral blood sample can include white blood cells, PBMC, etc. Any technology known in the art for separating white blood cells from a peripheral blood sample can be used. For example, a blood sample can be drawn, red blood cells can be lysed, and a white blood cell pellet can be isolated and used for the sample. In another example, density gradient separation can be used to separate white blood cells (such as PBMC) from red blood cells. In some embodiments, a fresh peripheral blood sample (that is, a sample not prepared by the above method) can be used. In some embodiments, a peripheral blood sample can be prepared by incubating in a solution to maintain the integrity of mRNA and / or protein.

[0153] In some embodiments, the responsiveness to therapy can refer to any one or more of the following: prolonging survival (including overall survival and progression-free survival); causing objective response (including complete response or partial response); or improving signs or symptoms of cancer. In some embodiments, responsiveness can refer to the improvement of one or more factors according to the published RECIST guideline group for determining tumor status in cancer patients, i.e., response, stability or progression. For a more detailed discussion of these guidelines, see Eisenhauer et al. (2009 Eur J Cancer 45: 228-47), Topalian et al. (2012 N Engl J Med 366: 2443-54), Wolchok et al. (2009 Clin Can Res 15: 7412-20) and Therasse et al. (2000 J. Natl. Cancer Inst. 92: 205-16). Responsive subject can refer to a subject whose cancer (one or more) shows improvement, for example, according to one or more factors based on RECIST criteria. A non-responsive subject can refer to a subject whose cancer(s) do not show improvement, for example, according to one or more factors based on RECIST criteria.

[0154] Conventional response criteria may not be sufficient to describe the anti-tumor activity of the therapeutic agent of the present invention, and it may produce a delayed response, which may occur after the initial obvious radiological progression (including the emergence of new lesions). Therefore, improved response criteria have been formulated, which take into account the new lesions that may appear and allow for confirmation of radiological progression in subsequent assessments. Accordingly, in some embodiments, responsiveness can refer to an improvement in one or more factors according to immune-related response criteria (irRC). See, for example, Wolchok et al. (2009, supra). In some embodiments, new lesions are added to the defined tumor burden and followed up in subsequent assessments, for example, for radiological progression. In some embodiments, the presence of non-target lesions is included in the assessment of complete response, but is not included in the assessment of radiological progression. In some embodiments, radiological progression can be determined based only on measurable disease and / or can be confirmed by continuous assessment of ≥4 weeks from the date of first recording.

[0155] In some embodiments, responsiveness can include immune activation. In some embodiments, responsiveness can include therapeutic efficacy. In some embodiments, responsiveness can include immune activation and therapeutic efficacy.

[0156] 3. Biomarker Panel

[0157] The biomarkers of the present invention can be used in predictive and / or prognostic tests to assess, determine and / or characterize (used interchangeably herein) the status of a therapy response signature in a patient, thereby guiding the patient's treatment. The phrase "status of a therapy response signature" includes a high therapy response signature (high RT) and a low therapy response signature (low RT). Based on this status, further procedures can be prescribed, including additional tests or treatment procedures or regimens.

[0158] The therapy response signature panel suitably includes one or more of EOMES-641K-Ac, EOMES-641K-Me, and / or EOMES-373K-Me. It will be appreciated that any one or more other biomarkers may also be included in the panel, for example, IFN-γ, TNF-α, IL-2, Ki67, PD-1, and / or CD107a.

[0159] The ability of an assay to correctly predict a response to a therapy is typically measured as the area under the curve of the assay's sensitivity, assay specificity, or receiver operating characteristic ("ROC"). Sensitivity refers to the percentage of true positives predicted as positive by the test, while specificity refers to the percentage of true negatives predicted as negative by the test. The ROC curve provides the sensitivity of a test as a function of 1-specificity. The greater the area under the ROC curve, the greater the predictive value of the test. Other useful metrics for test effectiveness are positive predictive value and negative predictive value. The positive predictive value is the percentage of people who test positive who are actually positive. The negative predictive value is the percentage of people who test negative who are actually negative.

[0160] In certain embodiments, the biomarker signatures of the present invention can show statistical differences in different treatment response states, at least p < 0.05, p < 10 -2 , p<10 -3 , p<10 -4 or p<10 -5 Predictive or prognostic tests using these biomarkers can demonstrate a ROC of at least 0.6, at least about 0.7, at least about 0.8, or at least about 0.9.

[0161] In certain embodiments, biomarkers in patient samples are measured using the methods described herein, and the state of the therapy response signature is calculated. In a specific embodiment, the measured value can then be compared with a relevant prediction or prognosis amount, cutoff value, or multivariate model score, which distinguishes a high therapy response signature (high RT) state from a low therapy response signature (low RT) state. The prediction or prognosis amount represents the measured amount of the biomarker (one or more), and according to being higher or lower than this amount, the patient is classified as having a state of a specific therapy response signature. As is well known in the art, by adjusting the specific prediction or prognosis cutoff value (one or more) used in the determination, the sensitivity or specificity of the assay can be improved according to the preference of the technician. In a specific embodiment, for example, the level or amount of the biomarker in a statistically significant number of samples from patients with different therapy response signature states can be measured, and a cutoff value suitable for the required specificity and sensitivity level can be obtained to determine a specific prediction or prognosis cutoff value.

[0162] Moreover, in certain embodiments, the biomarker measurements of the biomarker group are mathematically combined, and the combined value is associated with a potential prediction or prognosis problem of a high or low therapy response signature. The value of the biomarker can be combined by any appropriate mathematical method known in the art. The well-known mathematical method for associating the biomarker combination with the disease state adopts the following methods, such as discriminant analysis (DA) (for example, linear, quadratic, regularized DA), discriminant function analysis (DFA), Kernel method (for example, SVM), multidimensional scaling (MDS), nonparametric methods (for example, k- nearest neighbor classifier), PLS (partial least squares), tree-based methods (for example, logistic regression, CART, random forest algorithm, Boosting / Bagging method), generalized linear models (for example, logistic regression), principal component-based methods (for example, SIMCA), generalized additive models, fuzzy logic-based methods, methods based on neural networks and genetic algorithms. Those skilled in the art will not have problems selecting appropriate methods to evaluate the biomarker combination of the present invention. In one embodiment, the method used in correlating the biomarker combinations of the present invention is selected from DA (e.g., linear, quadratic, regularized discriminant analysis), DFA, Kernel methods (e.g., SVM), MDS, non-parametric methods (e.g., k-nearest neighbor classifier), PLS (partial least squares), tree-based methods (e.g., logistic regression, CART, random forest algorithm, Boosting method) or generalized linear models (e.g., logistic regression) and principal component analysis.For detailed information on these statistical methods, please see the following references: Ruczinski et al., 12 J. OF COMPUTATIONAL AND GRAPHICAL STATISTICS 475-511 (2003); Friedman, JH, 84 J. OF THE AMERICAN STATISTICAL ASSOCIATION 165-75 (1989); Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, The Elements of Statistical Learning, Springer Series in Statistics (2001); Breiman, L., Friedman, JH, Olshen, RA, Stone, CJ Classification and regression trees, California: Wadsworth (1984); Breiman, L., 45 MACHINE LEARNING 5-32 (2001); Pepe, MS, The Statistical Evaluation of Medical Tests for Classification and Prediction, Oxford Statistical Science Series, 28 (2003); and Duda, RO, Hart, PE, Stork, DG, Pattern Classification, Wiley Interscience, 2nd edition (2001).

[0163] 4. Generate a classification algorithm for qualitative therapy response signature status

[0164] In some embodiments, data generated using samples, such as "known samples," can be used to subsequently "train" a classification model. A "known sample" is a sample that has been pre-classified. The data used to form a classification model can be referred to as a "training data set." The training data set used to form a classification model can include raw data or pre-processed data. Once trained, the classification model can recognize patterns in the data generated using unknown samples. The classification model can then be used to classify the unknown samples into categories. For example, this can be useful in predicting whether a particular biological sample is associated with a particular biological condition.

[0165] Any suitable statistical classification or learning method can be used to form a classification model that attempts to separate a body of data into categories based on objective parameters present in the data. Classification methods can be supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition: A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, the teachings of which are incorporated herein by reference.

[0166] In supervised classification, training data containing examples of known classes is submitted to a learning mechanism that learns one or more sets of relationships that define each known class. New data can then be applied to the learning mechanism, which then uses the learned relationships to classify the new data. Examples of supervised classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression, and principal component regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART), artificial neural networks such as backpropagation networks, discriminant analysis (e.g., Bayesian classifiers or Fischer analysis), logistic classifiers, and support vector classifiers (SVMs).

[0167] Another supervised classification method is the recursive partitioning process. The recursive partitioning process uses a recursive partitioning tree to classify data derived from unknown samples. More detailed information about the recursive partitioning process is provided in U.S. patent application publication number 2002 0138208A1, inventors Paulse et al., entitled "Method for Analyzing Mass Spectra."

[0168] In other embodiments, the classification model created can be formed using unsupervised learning methods. Unsupervised classification attempts to learn classifications based on similarities in a training data set without pre-classifying the spectrum from which the training data set was derived. Unsupervised learning methods include cluster analysis. Cluster analysis attempts to divide data into "clusters" or groups, which ideally should have members that are very similar to each other and completely different from members of other clusters. Similarity is then measured using some distance metric that measures the distance between data items and clusters data items that are closer to each other. Clustering techniques include MacQueen's K-means algorithm and Kohonen's self-organizing map algorithm.

[0169] Learning algorithms disclosed for use in biological information classification are described, for example, in PCT International Application Publication No. WO 01 / 31580 (Barnhill et al., “Methods and devices for identifying patterns in biological systems and methods of use thereof”), U.S. Patent Application Publication No. 2002 / 0193950 (Gavin et al., “Method or analyzing mass spectra”), U.S. Patent Application Publication No. 2003 / 0004402 (Hitt et al., “Process for discriminating between biological states based on hidden patterns from biological data”), and U.S. Patent Application Publication No. 2003 / 0055615 (Zhang and Zhang, “Systems and methods for processing biological expression data”).

[0170] The classification model can be formed and used on any suitable digital computer. Suitable digital computers include micro, mini or mainframe computers using any standard or specialized operating system, such as those based on Unix, or Linux TM In embodiments employing a mass spectrometer, the digital computer used may be physically separate from the mass spectrometer used to create the spectrum of interest, or it may be coupled to the mass spectrometer.

[0171] The training data set and classification model according to the embodiments of the present invention can be implemented by a computer code executed or used by a digital computer. The computer code can be stored on any suitable computer readable medium, including optical or magnetic disks, memory sticks, magnetic tapes, etc., and can be written in any suitable computer programming language, including R, C, C++, etc. ++ , Visual Basic, etc.

[0172] The above learning algorithms are useful for developing classification algorithms for already discovered biomarkers as well as for finding new biomarkers. The classification algorithms then form the basis of diagnostic tests by providing diagnostic values ​​(eg, cutoff points) for the biomarkers used alone or in combination.

[0173] In some embodiments, any classification method disclosed herein can be at least partially executed by one or more computers and / or can be stored in a database on a non-transitory computer medium. In some embodiments, any classification method disclosed herein can be at least partially embodied or stored on a computer-readable medium having computer-executable instructions thereon. In some embodiments, a computer-readable medium includes any non-transitory and / or tangible computer-readable medium.

[0174] 5. Antibodies and Cell Lines

[0175] The present invention discloses the use of antigen binding molecules that specifically bind to these biomarkers to locate, detect and quantify therapeutic response biomarkers, particularly for EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me. Such antigen binding molecules are typically isolated acetylation or methylation site-specific antigen binding molecules that specifically bind to EOMES only when 641K is acetylated or methylated or when 373K is methylated. Using the acetylation and methylation site sequence information provided herein and as described in the examples, such antigen binding molecules can be produced by standard antibody production methods, such as anti-peptide antibody methods. For example, an antibody that specifically binds to EOMES-641K-Ac, EOMES-641K-Me or EOMES-373K-Me can be produced by immunizing an animal with a peptide antigen comprising all or part of the following amino acid sequence, which encompasses the corresponding acetylated or methylated residues (for example, a peptide antigen comprising the sequence shown in SEQ ID NO: 3, 4 or 5 (which encompasses acetylated or methylated lysine (suitably, dimethyllysine) at position 641 of EOMES and methylated lysine (suitably, dimethyllysine) at position 373 of EOMES)) to produce an antibody that binds to EOMES only when it is acetylated or methylated at position 641 or methylated at position 373.

[0176] The polyclonal antibodies of the present invention can be produced according to standard techniques by immunizing appropriate animals (e.g., rabbits, goats, etc.) with peptide antigens corresponding to the acetylation or methylation sites of the protein of interest, collecting immune serum from the animals, and isolating polyclonal antibodies from the immune serum according to standard procedures. For example, if it is desired to bind to an antibody of EOMES only when it is acetylated or methylated at 641K, the peptide antigen includes an acetylated or methylated form of lysine (e.g., K(Ac) or K(Me2) respectively). Conversely, if it is desired to bind to an antibody of EOMES only when it is not acetylated or methylated at 641K, the peptide antigen includes a conventional lysine form that is non-acetylated and non-methylated.

[0177] Peptide antigens suitable for generating antibodies of the present invention can be designed, constructed and used according to well-known techniques. See, for example, ANTIBODIES: A LABORATORY MANUAL, Chapter 5, pages 75-76, edited by Harlow & Lane, Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962).

[0178] Those skilled in the art will appreciate that longer or shorter acetylated or methylated peptide antigens may be used. For example, the peptide antigen may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, or 5, or it may comprise other amino acids flanking the sequence, or it may comprise only a portion of the disclosed sequence flanking an acetylated or methylated lysine. Typically, the desired peptide antigen will comprise four or more amino acids flanking each side of an acetylated or methylated amino acid and encompassing the acetylated or methylated amino acid. Polyclonal antibodies generated as described herein may be screened as further described below.

[0179] The monoclonal antibodies of the present invention can be produced in hybridoma cell lines according to the well-known techniques of Kohler and Milstein. See Nature 265:495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6:511 (1976); See also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, edited by Ausubel et al. (1989). The monoclonal antibodies thus produced have a high degree of specificity and improve the selectivity and specificity of the diagnostic assays provided by the present invention. For example, a solution containing an appropriate antigen can be injected into mice or other species, and then after a sufficiently long time (according to conventional techniques), the animals are sacrificed and spleen cells are obtained. Then, the spleen cells are immortalized by fusing them with myeloma cells, usually in the presence of polyethylene glycol, to produce hybridoma cells. For example, rabbit fusion hybridomas can be produced as described in U.S. Patent No. 5,675,063, C. Knight, authorized on October 7, 1997. The hybridoma cells are then grown in an appropriate selective medium, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant is screened for monoclonal antibodies with the desired specificity, as described below. The secreted antibodies can be recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange, or affinity chromatography.

[0180] Monoclonal Fab fragments can also be produced in E. coli by recombinant techniques known to those skilled in the art. See, for example, W. Huse, Science 246:1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87:8095 (1990). If a monoclonal antibody of one isotype is preferred for a particular application, then a specific isotype can be prepared directly by selection from the initial fusion, or by isolating class-switched variants using sib selection technology, secondary preparation from parent hybridomas that secrete monoclonal antibodies of different isotypes (Steplewski et al., Proc. Nat'l. Acad. Sci., 82:8653 (1985); Spira et al., J. Immunol. Methods, 74:307 (1984)).

[0181] The preferred epitope of the acetylation site-specific antibody or methylation site-specific antibody of the present invention is a peptide fragment consisting essentially of about 8 to 17 amino acids, including an acetylated or methylated lysine, wherein about 3 to 8 amino acids are located on each side of the acetylated lysine, so that the antibody of the present invention specifically binds to a post-translationally modified EOMES polypeptide comprising such an epitope sequence. A particularly preferred epitope bound by the antibody of the present invention comprises all or part of an acetylated or methylated site sequence, including acetylated or methylated amino acids.

[0182] Included within the scope of the present invention are equivalent non-antibody molecules such as antigen-binding fragments that bind to essentially the same acetyl or methyl-specific epitopes as those bound to the acetyl or methyl-specific antigen-binding molecules of the present invention in an acetyl or methyl-specific manner. See, for example, Neuberger et al., Nature 312:604 (1984). Such equivalent non-antibody reagents can be suitably used in the methods of the present invention described further below.

[0183] The antigen binding molecules encompassed by the present invention can be any type of antibody, including immunoglobulins, including IgG, IgM, IgA, IgD and IgE, and antigen binding fragments thereof. Antibodies can be monoclonal or polyclonal and can be of any species origin, including (for example) mouse, rat, rabbit, horse or human, or can be chimeric antibodies. See, for example, M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrison et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984). Antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Patent No. 4,474,893 (Reading) or U.S. Patent No. 4,816,567 (Cabilly et al.). Antibodies can also be chemically constructed by preparing specific antibodies according to the methods disclosed in US Pat. No. 4,676,980 (Segel et al.).

[0184] The present invention also provides immortalized cell lines that produce antibodies of the present invention. For example, hybridoma clones constructed as described above are also provided, which produce monoclonal antibodies against the EOMES acetylation or methylation sites disclosed herein. Similarly, the present invention includes recombinant cells that produce antibodies of the present invention, which can be constructed by well-known techniques; for example, the antigen binding site of the monoclonal antibody can be cloned by PCR, and the single-chain antibody produced is a recombinant antibody displayed on a phage or a soluble antibody in Escherichia coli (see, for example, ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, edited by Sudhir Paul).

[0185] The acetylated or methylated site-specific antibodies of the present invention, whether polyclonal or monoclonal, can be screened for epitopes and acetyl or methyl specificity according to standard techniques. See, for example, Czemik et al., Methods in Enzymology, 201: 264-283 (1991). For example, antibodies can be screened for acetyl and non-acetyl peptide libraries by ELISA to ensure specificity to the desired antigen and reactivity only to acetylated or methylated (or non-acetylated, non-methylated) forms of antigens. Peptide competition assays can be performed to confirm the lack of reactivity with other acetyl epitopes on a given protein acetylation signal transduction protein. Antibodies can also be tested by Western blotting for cell preparations containing signal transduction proteins (e.g., cell lines overexpressing target proteins) to confirm reactivity with desired acetylated epitopes / targets.

[0186] The specificity for the desired acetylated or methylated epitope can also be checked by constructing a mutant lacking an acetylated or methylated residue in a position outside the known desired epitope to be acetylated, or by mutating the desired acetylated or methylated epitope and confirming the lack of reactivity. The acetylated or methylated site-specific antigen binding molecules of the present invention may show some limited cross-reactivity to the relevant epitopes in non-target proteins. This is not surprising, because most antigen binding molecules show a certain degree of cross-reactivity, and anti-peptide antibodies often cross-react with epitopes with a high degree of homology to immunoreactive peptides. See, for example, Czemik, supra. It is easy to characterize with the cross-reactivity of non-target proteins by Western blotting and markers of known molecular weight. The amino acid sequence of the cross-reactive protein can be checked to identify a site with a high degree of homology to the EOMES epitope (antigen binding molecules of the present invention have specificity thereto).

[0187] In some cases, the polyclonal antiserum may show some undesirable general cross-reactivity to acetyl lysine or methyl lysine (suitably, dimethyl lysine) itself, which can be removed by further purification of the antiserum, for example, by purification on an acetyl tyramide or methyl tyramide column. The antigen binding molecules of the present invention specifically bind to EOMES only when acetylated or methylated at 641K or 373K (or only when not acetylated and not methylated, as the case may be), and do not (substantially not) bind to another form (compared to the form to which the antigen binding molecule is specific).

[0188] Antigen binding molecules can be further characterized via IHC or IF using normal and dysfunctional (e.g., exhausted) T cells to check EOMES acetylation or methylation. IHC or IF can be performed according to well-known technology. See, for example, ANTIBODIES:A LABORATORY MANUAL, Chapter 10, edited by Harlow & Lane, Cold Spring Harbor Laboratory (1988). For example, in brief, paraffin-embedded tissue (e.g., tumor tissue) is prepared for immunohistochemical staining, which is dewaxed with xylene to tissue sections and then treated with ethanol; first in water and then hydrated in PBS; by heating slides in sodium citrate buffer to expose antigens; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slides in primary and secondary antibodies; finally detecting using ABC avidin / biotin method according to manufacturer's instructions. For example, IF can be performed substantially as described in the following examples.

[0189] Antigen binding molecules can be further characterized by flow cytometry performed according to standard methods. See Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 7205-238 (2001). For example, the following protocol can be used for cytometry analysis: the sample can be centrifuged on a Ficoll gradient to remove red blood cells, and then the cells can be fixed with 2% paraformaldehyde for 10 minutes at 37°C, followed by permeabilization in 90% methanol on ice for 30 minutes. The cells can then be stained with an acetylated or methylated site-specific antigen binding molecule primary antibody (e.g., which detects EOMES-641K-Ac, EOMES-641K-Me or EOMES-373K-Me), washed and labeled with a fluorescently labeled secondary antibody. At this point, other fluorescent dye-conjugated biomarker antibodies (e.g., IFN-γ, TNF-α, IL-2, Ki67, PD-1 and / or CD107a) can also be added to assist in assessing T cell function. The cells can then be analyzed on a flow cytometer according to the specific protocol of the instrument used.

[0190] Antigen binding molecules can advantageously be conjugated to fluorescent dyes (eg, Alexa Fluor 488) for multiparameter analysis.

[0191] The acetylated or methylated site-specific antigen binding molecules of the present invention specifically bind to human EOMES polypeptides only when acetylated or methylated at 641K or methylated at 373K, but are not limited to binding to the human species itself. The present invention includes antigen binding molecules that, in addition to binding to human acetylation or methylation sites, also bind to conserved and highly homologous or identical acetylation or methylation sites in corresponding EOMES proteins from other species (e.g., mouse, rat, monkey, yeast). Highly homologous or identical conserved sites in other species can be easily identified by standard sequence comparison, for example, using BLAST identification using the human EOMES acetylation and methylation sites disclosed herein.

[0192] 6. Test kit

[0193] The present invention also extends to a kit, which is used to determine that biomarkers include the expression of therapy response biomarkers disclosed herein and other biomarkers, which includes reagents allowing detection and / or quantification of biomarkers. Such reagents include, for example, compounds or materials or compound groups or material groups, which allow biomarkers to be quantified. In a specific embodiment, compounds, materials or compound groups or material groups allow determination of protein expression levels or gene expression levels, including but not limited to antigen binding molecules (such as antibodies), materials for extracting RNA, primers for synthesizing corresponding cDNA, primers for amplifying DNA, and / or probes capable of specific hybridization with RNA (or corresponding cDNA) encoded by genes, TaqMan probes, etc.

[0194] The kit may also optionally include suitable reagents for detecting the label, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, etc. For example, a protein-based detection kit may include (i) at least one EOMES polypeptide, suitably selected from EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me, or a fragment thereof comprising 641K-Ac, 641K-Me or 373K-Me, and an unacetylated or methylated EOMES polypeptide (which may be used as a control), and (ii) one or more antigen binding molecules that specifically bind to an EOMES polypeptide (e.g., EOMES-641K-Ac, EOMES-641K-Me and / or EOMES-373K-Me, and / or an unacetylated or methylated EOMES polypeptide). The antigen binding molecule is suitably detectably labeled. The kit can also have various devices (e.g., one or more) and reagents (e.g., one or more) for performing one of the assays described herein; and / or printed instructional materials for using the kit to quantify the expression of T cell function biomarker genes.

[0195] Materials suitable for packaging diagnostic kit components can include crystal glass, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, bags, etc. In addition, the kit of the present invention can include instructional materials for using the different components contained in the kit simultaneously, sequentially, or separately. The instructional materials can be in the form of printed materials or in the form of electronic carriers capable of storing instructions so that they can be read by the subject, such as electronic storage media (disks, tapes, etc.), optical media (CD-ROMs, DVDs, etc.). Alternatively or additionally, the medium can include an Internet address that provides the instructional materials.

[0196] 7. Patient classification and treatment management

[0197] The present invention extends to methods for selecting or identifying which individuals are suitable treatment candidates for the treatment of cancer using therapy (e.g., cytotoxic therapy, immunotherapy, etc.). Such individuals include patients predicted to respond to therapy, so relative to other patients with different characteristics (one or more) (e.g., no responsiveness to therapy), the possibility of benefiting from administering therapy increases. In certain embodiments, suitable candidates are candidates with reasonable likelihood of benefiting from treatment or at least having enough likelihood of benefiting so as to have reason to administer treatment considering its risks and side effects. The present invention also includes methods for selecting or identifying which individuals are not suitable treatment candidates for the treatment of cancer using therapy (e.g., cytotoxic therapy, immunotherapy, etc.). Such individuals include patients predicted to respond to therapy or have a weak response, so relative to other patients with different characteristics (one or more) (e.g., responsiveness to therapy), the possibility of benefiting from administering the therapy decreases, or the possibility of benefiting from such treatment is low or substantially absent, making it possible to expect to use different or other treatments. In some embodiments, the determination of at least one therapy response biomarker in a sample obtained from a subject determines whether the subject is a suitable candidate for treatment with therapy.

[0198] In some aspects, described herein is the possibility of determining that a subject in need of cancer treatment responds to therapy (e.g., cytotoxic therapy, immunotherapy, etc.), and / or identifying and / or selecting a method for a subject to receive such treatment, for example, based on a determination of at least one therapy response biomarker. In a specific embodiment, therapy is an immunotherapy, appropriately using anti-immune checkpoint inhibitors. The phrase "treated with immune checkpoint inhibitors", also referred to as "immune checkpoint inhibitor treatment", "therapy using immune checkpoint inhibitors" or "immune checkpoint inhibitor therapy", includes embodiments involving treatment with a single immune checkpoint inhibitor and embodiments involving combined treatment with two or more immune checkpoint inhibitors. In some embodiments, immune checkpoint inhibitor treatment includes the use of a single agent or two or more separate agents to suppress two or more different immune checkpoint pathways.

[0199] The invention also includes the use of methods for assessing T cell function or immune function in a subject as described herein in methods for selecting or identifying individuals with impaired or decreased immune function for treatment with therapies that stimulate or enhance immune function (e.g., immunotherapy, such as adoptive immunotherapy).

[0200] In order that the present invention may be readily understood and put into practical effect, certain preferred embodiments will now be described by way of the following non-limiting examples.

[0201] Example

[0202] Example 1

[0203] Expression of EOMES in T cells

[0204] To quantify CD8 + Functional properties of T cells were obtained from liquid biopsies of melanoma patients, metastatic breast cancer patients and healthy individuals stratified according to RECIST1.1 (which classifies responses to therapy based on changes in tumor mass; Eisenhauer et al., Eur J Cancer. 2009, 45(2): 228-47). Liquid biopsies are an important tool for quantifying the phenotype of immune cells in the blood. Patient subgroups included patients who had complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). In brief, CD8 T cells were isolated from liquid biopsies of healthy donors (HD), metastatic breast cancer patients or melanoma patients every 3 months within 24 months after baseline blood collection. + Melanoma patients were further stratified into complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) based on objective response to immunotherapy treatment (mono- or dual-therapy with pembrolizumab, nivolumab, and / or ipilimumab).

[0205] In stimulating CD8 + T cells were analyzed before and after stimulation with expression of effector markers and protein expression was analyzed by high-resolution immunofluorescence. + T cells cannot express effector proteins (Huang et al., 2017, Nature, 545(7652), 60-65; Wherry and Kurachi 2015, Nat Rev Immunol, 15(8), 486-499; Bengsch et al., 2018, Immunity, 48(5), 1029-1045; Catakovic et al., 2017, Cell Commun Signal, 15(1), 1; Woroniecka et al., 2018 Clin Cancer Res, 24(17), 4175-4186). Analysis of T cells in this study showed that the expression of Ki67, TNF-α and IFN-γ in the PD cohort was lower than that in the CR and SD melanoma cohorts, and no effect was observed after stimulation ( Figure 1 A).

[0206] Next, CD8 +T cells were probed for expression of the proposed exhaustion signature, PD-1 and EOMES. PD-1 was included because it is the primary inhibitory checkpoint displayed in dysfunctional T cells. The analysis showed that CD8 + The CD8 T cells of HD, CR and PR groups were higher than those of + T cells have significantly and prominently higher nuclear EOMES expression ( Figure 1 B).

[0207] Due to the importance of EOMES in exhausted, dysfunctional T cells, the EOMES sequence was examined to identify putative nuclear localization signals using NLS Mapper and online tools for identifying potential methylated residues. Sequences at the C-terminus were identified as having a high probability of mediating nuclear localization ( Figure 1 C) This region was identified as 635 VYTSACKRRRLSP 647 , and based on the prediction of species-specific methylation sites and acetylation sites, lysine (K) at position 641 (641K) was identified as a potential target for methylation / demethylation and a target for acetylation (Wen et al., 2016, Bioinformatics, 32(20), 3107-3115). Many studies have demonstrated the importance of various post-translational modifications in controlling protein-target interactions and localization. Therefore, it is speculated that methylation, demethylation, and acetylation of this lysine within the EOMES NLS may be critical in regulating its nuclear localization and dynamics.

[0208] To understand the significance of the post-translational modification of EOMES at 641 K, three plasmid constructs were prepared ( Figure 1 D). E-WT, representing the wild-type EOMES sequence; E-MUT1, in which 641K is mutated to arginine (R), mimicking lysine in its unmethylated, unacetylated state; and E-MUT2, in which 641K is mutated to phenylalanine, mimicking the hypermethylated state of lysine. Since E-MUT1 cannot be methylated or acetylated, this construct was used to demonstrate the importance of acetylation and methylation in nuclear entry (assuming that this mutation would alter nuclear entry kinetics). Mutations in E-MUT2 should render the EOMES polypeptide incapable of nuclear localization.

[0209] It was observed that the functional lysine at position 641 that can be demethylated is required for the nuclear translocation of EOMES to the nucleus, and that hypermethylation of EOMES at position 641K leads to a decrease in nuclear EOMES. Specifically, the effects of transfection of Jurkat cells with E-WT, E-MUT1, and E-MUT2 on EOMES, TBET, and PD-1 were examined. Figure 1 As shown in E. High-resolution microscopy revealed that, compared with E-WT, the localization of EOMES in cells transfected with E-MUT1 or E-MUT2 was significantly more biased towards the cytoplasm, while the nuclear EOMES was significantly reduced and the nuclear bias was also lower (as expressed by the fluorescence ratio of the nucleus to the cytoplasm: Fn / c) ( Figure 1 E) Compared to the vector-only control, EOMES expression in cells transfected with E-MUT1 was slightly, but not significantly, lower, while EOMES Fn / c remained cytoplasmic compared to the vector-only control. In cells transfected with E-MUT2, mean NFI and EOMES Fn / c were significantly lower than those observed in the vector-only control. TBET expression was slightly increased by transfection with E-WT, while transfection with E-MUT1 or E-MUT2 induced higher nuclear TBET expression compared to the vector-only control and E-WT. PD-1 expression was unaffected by transfection with E-WT, while both E-MUT1 and E-MUT2 transfections significantly attenuated PD-1 expression.

[0210] These data indicate that constitutive methylation of 641K (represented by E-MUT2) has different effects on cellular localization and protein expression than unmethylated / unacetylated 641K (represented by E-MUT1). This may be because methylation at 641K may affect protein interactions of EOMES, leading to different interactions and different effects on protein expression. This indicates the importance of demethylation of 641K in influencing the nuclear targeting and protein:protein interactions of EOMES.

[0211] As described above, the effects of transfection with E-WT, E-MUT1, and E-MUT2 on the expression of Ki67, IFN-γ, and TNF-α were examined in the same set of transfected Jurkat cells. High-resolution microscopy analysis showed that transfection with E-WT significantly attenuated the expression of Ki67, IFN-γ, and TNF-α compared to the vector-only control or E-WT. Transfection with E-MUT1 induced a significant increase in the expression of Ki67 and IFN-γ, but not TNF-α, relative to the control, however, the expression of these proteins was significantly increased when compared to E-WT transfected cells. Transfection with E-MUT2 had a significant and strong effect on the expression of all three proteins, with the observed expression being much higher than that in cells transfected with the control, E-WT, or E-MUT1 ( Figure 1 F).

[0212] These data indicate that nuclear localization of EOMES is necessary for attenuating the expression of effector markers, as shown by the attenuation of Ki67, IFN-γ, and TNF-α by E-WT. However, hypermethylated EOMES was also found to induce strong expression of Ki67, IFN-γ, and TNF-α. This suggests that different post-translational modifications of EOMES at key residues, in addition to their effects on localization, can also induce different protein interactions, as evidenced by the differences in effector protein expression induced by E-MUT1 and E-MUT2. It can therefore be concluded that the specific post-translational modification present at 641K in EOMES is important for regulating EOMES protein targets, as well as protein:target interactions and nuclear localization.

[0213] Example 2

[0214] Antibodies specific for post-translationally modified EOMES can predict responsiveness to therapy

[0215] To investigate the role of the 641K post-translational modification in the EOMES polypeptide, rabbit polyclonal antibodies specific for the NLS motif were generated against 641K methylated and 641K acetylated EOMES proteins (ie, EOMES-641K-Me and EOMES-641K-Ac). Figure 2 The specificity of the antibodies is shown.

[0216] In addition to the identified EOMES NLS, a new NLS located in The EOMES DNA binding domain, of which the central lysine at position 373 (373K) is critical for controlling target binding specificity. Based on the X-ray structure of the DNA binding domain within the transcription factor T-bet, a homology model of the EOMES:DNA complex was generated to assess potential interaction regions between EOMES and DNA / chromatin / promoter regions. The high sequence identity (72%) between EOMES and T-BET within the DNA binding domain provided a 100% confidence level in the model, and the coverage of EOMES and T-BET confirmed the high similarity of these regions. The lysine at position 373 in EOMES is conserved in T-BET (373K), which has been shown to associate with a phosphate group on DNA. Methylation of this lysine residue is predicted to interfere with DNA binding ( Figure 3 A rabbit polyclonal antibody was also generated against EOMES containing this residue methylated (i.e., EOMES-373K-Me) ( Figure 2 and 3 B).

[0217] The present inventors hypothesized that acetylation of EOMES-641K is activated in CD8 + It is also predicted to be more prevalent in the more responsive CD8 + EOMES-641K-Me would be more prevalent in T cells, as would EOMES-373K-Me. Therefore, the profiles of EOMES-641K-Me and EOMES-641K-Ac were examined in baseline formalin-fixed, paraffin-embedded (FFPE) tissue taken from two cohorts of melanoma patients (those who were classified as responders or resistant to immunotherapy after the start of treatment). Baseline FFPE tissue was collected before the start of treatment. Analysis of protein expression by high-resolution imaging showed that EOMES-641K-Me was present in T cells in both cohorts, but the levels in the responder cohort were significantly higher than those in the resistant cohort. The resistant cohort was the only cohort in which significant intensity of EOMES-641K-Ac was observed, while almost no EOMES-641K-Ac was observed in the responder cohort ( Figure 3 C; representative images not shown).

[0218] The relative presence of EOMES-641K-Me and EOMES-641K-Ac in baseline tissues may represent the underlying exhaustion characteristics of the patient sample and its ability to respond to immunotherapy alone. If the prevalence of EOMES-641K-Ac is high, this suggests that immunotherapy alone is insufficient and that additional treatment modalities, such as epigenetic drugs targeting post-translational modifications of EOMES-641K-Ac, may be needed.

[0219] High-resolution microscopy was then used to characterize CD8 + T cell prevalence of EOMES-641K-Ac was examined. Analysis showed that only CD8 + T cells have any significant level of EOMES-641K-Ac, and this EOMES-641K-Ac is also mainly localized to the nucleus ( Figure 3 D; representative images not shown).

[0220] CD8 T cells from healthy donors, patients who responded to immunotherapy, or patients who were resistant to immunotherapy were also characterized by high-resolution microscopy. + T cell analysis of EOMES-641K-Me profile. Analysis showed that CD8 +EOMES-641K-Me levels in T cells were significantly higher than those in the resistant and refractory patient cohorts or the healthy donor cohort. In all three cohorts, EOMES-641K-Me was mainly localized in the cytoplasm and only in CD8 T cells derived from responders. + Significant EOMES-641K-Me nuclear expression was detected in T cells ( Figure 3 E; representative images not shown).

[0221] CD8 from the same patient cohort + The prevalence of EOMES-373K-Me in T cells was also assessed by high-resolution microscopy. + The levels of EOMES-373K-Me in T cells were significantly higher than those in the resistant and refractory patient cohorts and, to a lesser extent, higher than those in the healthy donor cohort. It was also found that only in the responder cohort, EOMES-373K-Me was mainly localized to the nucleus ( Figure 3 F; representative images not shown).

[0222] CD8 TNBC + Analysis of EOMES-641K-Me and EOMES-641K-Ac in T cells showed that these cells also had CD8 T cells from resistant melanoma patients. + T cells matched the EOMES signature, suggesting that these T cells from TNBC patients were also exhausted ( Figure 3 G; representative images not shown). Notably, TNBC responds poorly to immunotherapy.

[0223] In conclusion, EOMES-641K-Ac was associated with patients who did not respond to immunotherapy and could therefore predict or classify patients as immunotherapy non-responders. In contrast, EOMES-641K-Me and EOMES-373K-Me were associated with patients who responded to immunotherapy and could therefore predict or classify patients as immunotherapy responders.

[0224] The data suggest that nuclear localization of EOMES is important for the exhaustion phenotype, maintaining its acetylated form and demethylation at positions 641 and 373. For effector function, a state seen in healthy T cells, EOMES is methylated and expressed in the cytoplasm, although it is also present in the nucleus.

[0225] The data also suggest a new role for EOMES. Depending on the presence of specific post-translational modifications at 641K and 373K, this factor can have both positive and negative functions. The nuclear localization of EOMES is also affected by the post-translational modification at 641K, and EOMES-641K-Me is found in CD8 + It is exclusively cytoplasmic in T cells but is also localized to the nucleus in cells from patients who respond to therapy.

[0226] Example 3

[0227] CD8 from patients with metastatic brain cancer + EOMES localization in T cells

[0228] FFPE tissues from metastatic brain lesions of patients with metastatic brain cancer were examined by the automated ASI mIF system targeting infiltrating CD8 + Interestingly, the analysis found that CD8 T cells expressing EOMES-641K-Ac in tumor lesions + CD8 T cells + Approximately 60% of the T cell population, while CD8 + T cells only account for CD8 + T cell population is less than 18%. + The expression intensity of EOMES-641K-Ac in T cells was significantly higher than that of EOMES-641K-Me. This indicates that in brain metastasis, CD8 + EOMES-641K-Ac is upregulated in T cells, indicating exhausted CD8 + T cell signature. ( Figure 4 ; representative images not shown).

[0229] Example 4

[0230] Materials and methods

[0231] Isolation of CD8 + T cells

[0232] Using RosetteSep TM Methods Metastatic melanoma biopsies were pre-enriched to isolate CD8 + T cells. RosetteSep TM Human CD8 enrichment kit (15063, Stemcell Technologies) was used to isolate CD8 +T cells and red blood cell removal with SepMate TM -50 (IVD) density gradient tubes (85450, Stemcell Technologies) and Lymphoprep TM Density gradient centrifugation was performed using density gradient medium (07861, Stemcell Technologies).

[0233] Immunofluorescence microscopy

[0234] The isolated CD8 + T cells or Jurkat cells were centrifuged onto coverslips pretreated with poly-l-lysine and fixed, and then stored in PBS for staining. Permeabilization cells were performed by incubation with 1% Triton X-100 for 20 minutes, and cells were detected using relevant antibodies (including anti-CD8, anti-TNF, anti-IFN-γ, anti-Ki67, anti-PD1, anti-EOMES, anti-TBET and anti-cytokeratin antibodies). Customized polyclonal rabbit anti-EOMES-641K-Ac, anti-EOMES-641K-Me and anti-EOMES-373K-Me were also used. Primary antibody was visualized using a secondary antibody conjugated with Alexa Fluor 488 (anti-rabbit), 568 (anti-mouse) or 647 (anti-rat).

[0235] FFPE samples from metastatic tumor biopsies were processed for OPAL staining (Perkin-Elmer) using the BOND RX instrument protocol: ER2 was performed at 100°C for 20 minutes using Epitope Retrieval Solution 1 (EDTA-based, pH 6.0), followed by probing with rabbit anti-EOMES-641K-Ac or anti-EOMES-641K-Me and mouse host CD8, and visualization with Opal kits 520, 570, and 690.

[0236] Coverslips were mounted on microscope slides using ProLong Diamond Antifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5-μm fields were acquired using a Leica DMI8 microscope with a 100× oil-immersion lens and LAX software. Final images were obtained by averaging four consecutive images of the same area. Digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA) to determine total fluorescence intensity (TFI), nuclear fluorescence intensity (NFI), and cytoplasmic fluorescence intensity (CFI). The Mann–Whitney nonparametric test (GraphPad Prism, GraphPad Software, San Diego, CA) was used to determine significant differences between data sets.

[0237] Production of EOMES antibodies

[0238] Antibodies were raised against the following peptides: EOMES NLS: VTYSCKRRRLSP (SEQ ID NO: 2); EOMES-641K-Ac: VTYSCK(Ac)RRRLSP (SEQ ID NO: 3); EOMES-641K-Me: VTYSCK(Me)RRRLSP (SEQ ID NO: 4); and EOMES-373K-Me: RQUISFGKLK(Me)LTNNKGANN (SEQ ID NO: 5). Since short peptides are generally not immunogenic on their own, they often need to be conjugated to an immunogenic carrier protein. To facilitate this conjugation, a cysteine ​​was incorporated into the C-terminus of the above peptide sequence and the peptide was conjugated to the immunogenic carrier protein, keyhole limpet hemocyanin (KLH). No special immunization protocol is required to generate antibodies against dimethylated or acetylated peptides. Two or four rabbits were immunized several weeks apart for each peptide sequence. The first immunization was with an emulsion of the peptide conjugate in complete Freund's adjuvant, and the second immunization was with incomplete Freund's adjuvant. Effective anti-peptide serum was obtained several weeks later (see Palfreyman et al. (1984) J Immunol Meth, 75:383).

[0239] Testing of antisera against dimethylated and acetylated peptides was performed using an enzyme-linked immunosorbent assay (ELISA) in which sera were titrated on microtiter plates coated with either non-post-translationally modified peptides, dimethylated peptides, or acetylated peptides.

[0240] Antibody enhancement was performed by coupling the unmodified peptide to the gel using existing cysteine ​​residues using SulfoLink Coupling Resin (Thermo Scientific, Product No. 20401) according to the manufacturer's instructions. The resulting gel was incubated with an aliquot of antiserum to adsorb antibodies specific for the unmodified peptide. The resulting antiserum had enhanced specificity for dimethylated or acetylated peptide sequences.

[0241] To generate affinity-purified antibodies specific only for dimethylated or acetylated peptides, an enhancement procedure was first performed to remove antibodies specific for the unmodified peptide from the serum. The specificity of the affinity-purified antibodies was tested by ELISA. Figure 2 As shown, the generated antibodies showed high specificity for various forms of the peptide.

[0242] ASI systems approach (high-throughput, high-resolution microscopy)

[0243] ASI's mIF system is a versatile scanning and analysis system for multiplexed immunofluorescence samples. It is designed to scan slides stained with DAPI and up to six antibodies, remove autofluorescence, resolve filter misintermixing, and perform cell-based analysis of the acquired data. It automatically segments touching cells, quantitatively measures signal expression, and displays results for each cell and the entire scan area. It supports a variety of automated and semi-automated scanning modes, including:

[0244] 1. Efficient density-based scanning of suspension samples - Scan samples based on cell populations for the fastest cell scoring;

[0245] 2. Scan the selected area / range; and

[0246] 3. Interactively scan specific locations of interest.

[0247] In all modes, the comprehensive statistics of tens of thousands of cells with antibody co-localization can be derived in a few minutes. 3D stacking, automatic exposure, autofocus and other imaging parameters are inherent parts of each scan. The image is used to determine the average nuclear fluorescence intensity (NFI) or overall fluorescence intensity (FI). Using an automatic platform and ASI software (for automatically selecting cells and measuring fluorescence intensity), the total number of cells is counted in the defined area. Subsequently, the resulting data is used to calculate the CTC group dynamics, expressed as the % of the total cell population.

[0248] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0249] The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" to the present application.

[0250] Throughout this specification, the purpose has been to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or particular set of features. Therefore, those skilled in the art will appreciate that various modifications and variations may be made to the specific embodiments recited in light of this disclosure without departing from the scope of the present invention. All such modifications and variations are intended to be included within the scope of the appended claims.

Claims

1. Use of a reagent for detecting post-translational modifications in the nuclear localization sequence and / or DNA binding motif of EOMES in T cells in the preparation of a preparation for a method for evaluating T cell function, wherein the post-translational modification in the nuclear localization sequence is selected from EOMES-641K-Ac and EOMES-641K-Me, and wherein the post-translational modification in the DNA binding motif is EOMES-373K-Me.

2. The method of claim 1, wherein the method comprises detecting acetylation of EOMES-641K, i.e., EOMES-641K-Ac, in T cells and determining T cell dysfunction.

3. The use of claim 2, wherein the method comprises detecting an increased level of EOMES-641K-Ac in T cells relative to a suitable control.

4. The use according to claim 2 or 3, wherein the method further comprises detecting the cellular localization of EOMES-641K-Ac in T cells.

5. The use of claim 4, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-641K-Ac in T cells.

6. The use according to claim 5, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Ac in T cells, or the ratio of cytoplasmic to nuclear localization.

7. The use of claim 1, wherein the method comprises detecting methylation of EOMES-641K, i.e., EOMES-641K-Me, in T cells and determining that the T cells are functional.

8. The use of claim 7, wherein the method comprises detecting elevated levels of EOMES-641K-Me in T cells relative to a suitable control.

9. The use according to claim 7 or 8, wherein the method further comprises detecting the cellular localization of EOMES-641K-Me in T cells.

10. The use of claim 9, wherein the method comprises detecting the cytoplasmic and / or nuclear localization of EOMES-641K-Ac in T cells.

11. The use of claim 10, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Me in T cells, or the ratio of cytoplasmic to nuclear localization.

12. The use of claim 1, wherein the method comprises detecting methylation of EOMES-373K (EOMES-373K-Me) in T cells and determining that the T cells are functional.

13. The use of claim 12, wherein the method comprises detecting elevated levels of EOMES-373K-Me in T cells relative to a suitable control.

14. The use according to claim 12 or 13, wherein the method further comprises detecting the cellular localization of EOMES-373K-Me in T cells.

15. The use of claim 14, wherein the method comprises detecting the cytoplasmic and / or nuclear localization of EOMES-373K-Me in T cells.

16. The use according to claim 15, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-373K-Me in T cells, or the ratio of cytoplasmic to nuclear localization.

17. Use of an agent for detecting post-translational modifications in the nuclear localization sequence of EOMES and / or the DNA binding motif of EOMES in a T cell or T cell population in the preparation of a preparation for a method for predicting the likelihood of a subject with cancer to respond to therapy, wherein the post-translational modification in the nuclear localization sequence is selected from EOMES-641K-Ac and EOMES-641K-Me, and wherein the post-translational modification in the DNA binding motif is EOMES-373K-Me.

18. The use of claim 17, wherein the method comprises detecting acetylation of EOMES-641K in a T cell or a population of T cells, thereby determining that the subject has an increased likelihood of being resistant or unresponsive to the therapy.

19. The use of claim 18, wherein the method comprises detecting an increased level of EOMES-641K-Ac in a T cell or T cell population relative to a suitable control.

20. The use of claim 18 or 19, wherein the method further comprises detecting the cellular localization of EOMES-641K-Ac in T cells.

21. The use of claim 20, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-641K-Ac in T cells.

22. The use of claim 21, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Ac in T cells, or the ratio of cytoplasmic to nuclear localization.

23. The use of claim 17, wherein the method comprises detecting methylation of EOMES-641K in a T cell or a population of T cells, thereby determining that the subject has an increased likelihood of being sensitive or responsive to the therapy.

24. The use of claim 23, wherein the method comprises detecting an increased level of EOMES-641K-Me in a T cell or T cell population relative to a suitable control.

25. The use of claim 23 or 24, wherein the method further comprises detecting the cellular localization of EOMES-641K-Me in T cells.

26. The use of claim 25, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-641K-Me in T cells.

27. The use of claim 26, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-641K-Me in T cells, or the ratio of cytoplasmic to nuclear localization.

28. The use of claim 17, wherein the method comprises detecting methylation of EOMES-373K in a T cell or a population of T cells, thereby determining that the subject has an increased likelihood of being sensitive or responsive to the therapy.

29. The use of claim 28, wherein the method comprises detecting an increased level of EOMES-373K-Me in a T cell or T cell population relative to a suitable control.

30. The use of claim 28 or 29, wherein the method further comprises detecting the cellular localization of EOMES-373K-Me in T cells.

31. The use of claim 30, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-373K-Me in T cells.

32. The use of claim 31, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization of EOMES-373K-Me in T cells, or the ratio of cytoplasmic to nuclear localization.

33. Use of a reagent for detecting the presence of EOMES-641K-Ac in a T cell or a population of T cells for the preparation of a preparation for use in a method of determining the likelihood that a subject with cancer is resistant to therapy.

34. The use of claim 33, wherein the method comprises detecting elevated levels of EOMES-641K-Ac in a T cell or T cell population relative to a suitable control, which indicates that the subject has an increased likelihood of being resistant to therapy.

35. The use of claim 33 or 34, wherein the method comprises contacting a sample comprising T cells or a population of T cells with an antigen binding molecule that specifically binds to EOMES-641K-Ac, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Ac in the sample, thereby determining that the subject has an increased likelihood of being resistant to therapy.

36. Use of a reagent for detecting the presence of EOMES-641K-Me in a T cell or a population of T cells for the preparation of a preparation for use in a method for determining the likelihood that a subject suffering from cancer is sensitive to a therapy.

37. The use of claim 36, wherein the method comprises detecting an elevated level of EOMES-641K-Me in a T cell or T cell population relative to a suitable control, which indicates that the subject has an increased likelihood of being sensitive to the therapy.

38. The use of claim 36 or 37, wherein the method comprises contacting a sample comprising T cells or a population of T cells with an antigen binding molecule that specifically binds to EOMES-641K-Me, and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby determining that the subject has an increased likelihood of sensitivity to the therapy.

39. Use of a reagent for detecting the presence of EOMES-373K-Me in a T cell or a population of T cells for the preparation of a preparation for use in a method for determining the likelihood that a subject suffering from cancer is sensitive to a therapy.

40. The use of claim 39, wherein the method comprises detecting an elevated level of EOMES-373K-Me in a T cell or T cell population relative to a suitable control, which indicates that the subject has an increased likelihood of being sensitive to the therapy.

41. The use of claim 39 or 40, wherein the method comprises contacting a sample comprising T cells or a population of T cells with an antigen binding molecule that specifically binds to EOMES-373K-Me, and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby determining that the subject has an increased likelihood of sensitivity to the therapy.

42. Use of a reagent for measuring the level of EOMES-641K-Ac and a reagent for measuring the level of EOMES-641K-Me in a T cell or T cell population in the preparation of a preparation for use in a method for predicting the likelihood of response to therapy in a subject with cancer, wherein the method comprises: The levels of EOMES-641K-Ac and EOMES-641K-Me in T cells or T cell populations were compared; and predicting the subject's response to the therapy based on the comparison, wherein a higher level of EOMES-641K-Ac than EOMES-641K-Me indicates an increased likelihood that the subject is resistant to the therapy, and wherein a higher level of EOMES-641K-Me than EOMES-641K-Ac indicates an increased likelihood that the subject is sensitive to the therapy.

43. The use of claim 42, wherein the method comprises: A sample comprising T cells or a population of T cells is contacted with a first antigen-binding molecule that specifically binds to EOMES-641K-Ac and a second antigen-binding molecule that specifically binds to EOMES-641K-Me; the level of a first complex comprising the first antigen-binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen-binding molecule and EOMES-641K-Me are measured in the sample; and the likelihood of the subject responding to the therapy is predicted based on the comparison, wherein a higher level of the first complex than the second complex in the sample indicates an increased likelihood that the subject is resistant to the therapy, and wherein a higher level of the second complex in the sample indicates an increased likelihood that the subject is sensitive to the therapy.

44. The use of any one of claims 1-3, 5-8, 10-13, 15-19, 21-24, 27-29, 31-34, 36, 37, 39, 40, 42, and 43, wherein the method further comprises detecting at least one additional biomarker in the T cell or T cell population.

45. The use of claim 44, wherein the at least one additional biomarker is selected from IFN-γ, TNF-α, IL-2, Ki67, PD-1, and CD107a.

46. ​​Use of a reagent for detecting post-translational modifications in the nuclear localization sequence and / or DNA binding motif of EOMES in a T cell or T cell population in the preparation of a preparation for a method for stratifying subjects with cancer into possible responders or non-responders to therapy, wherein the post-translational modification in the nuclear localization sequence is selected from EOMES-641K-Ac and EOMES-641K-Me, and wherein the post-translational modification in the DNA binding motif is EOMES-373K-Me.

47. The use of claim 46, wherein the method comprises detecting EOMES-641K-Ac in a T cell or T cell population and stratifying the subject as a likely non-responder to the therapy.

48. The use of claim 47, wherein the method comprises contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Ac and detecting a complex comprising the antigen binding molecule and EOMES-641K-Ac in the sample, thereby stratifying the subject into a potential non-responder to the therapy.

49. The use of claim 48, wherein the method comprises detecting EOMES-641K-Me in a T cell or T cell population and stratifying the subject into a likely responder to the therapy.

50. The use of claim 49, wherein the method comprises contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Me and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby stratifying the subject into a likely responder to the therapy.

51. The use of claim 46, wherein the method comprises detecting EOMES-373K-Me in a T cell or T cell population and stratifying the subject into a likely responder to the therapy.

52. The use of claim 51, wherein the method comprises contacting the sample with an antigen binding molecule that specifically binds to EOMES-373K-Me and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby stratifying the subject into a possible responder to the therapy.

53. The use of any one of claims 46-50, wherein the method comprises contacting a sample with a first antigen binding molecule that specifically binds to EOMES-641K-Ac and a second antigen binding molecule that specifically binds to EOMES-641K-Me; measuring the level of a first complex comprising the first antigen binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen binding molecule and EOMES-641K-Me in the sample; and stratifying the subject into a possible responder or a non-responder based on a comparison of the level of the first complex with the level of the second complex, wherein in the sample, if the level of the first complex is higher than the level of the second complex, the subject is stratified as a possible non-responder, and wherein if the level of the second complex is higher than the level of the first complex, the subject is stratified as a possible responder.

54. Use of an agent for detecting a post-translational modification in the nuclear localization sequence and / or DNA binding motif of EOMES in a T cell or a population of T cells in the preparation of a preparation for use in a method of managing a subject with cancer with a therapy, wherein the method comprises: Selecting a subject with cancer to be treated with the therapy based on the subject being a likely responder to the therapy, or selecting a subject with cancer not to be treated with the therapy based on the subject being a likely non-responder to the therapy, and treating the subject with or without the therapy based on the selection, wherein the post-translational modification in the nuclear localization sequence is selected from EOMES-641K-Ac and EOMES-641K-Me, and wherein the post-translational modification in the DNA binding motif is EOMES-373K-Me.

55. The use of claim 54, wherein the method comprises detecting EOMES-641K-Me in a T cell or T cell population and stratifying the subject as a likely responder to the therapy.

56. The use of claim 55, wherein the method comprises contacting the sample with an antigen binding molecule that specifically binds to EOMES-641K-Me and detecting a complex comprising the antigen binding molecule and EOMES-641K-Me in the sample, thereby stratifying the subject into a likely responder to the therapy.

57. The use of claim 54, wherein the method comprises detecting EOMES-373K-Me in a T cell or T cell population and stratifying the subject into a likely responder to the therapy.

58. The use of claim 57, wherein the method comprises contacting the sample with an antigen binding molecule that specifically binds to EOMES-373K-Me and detecting a complex comprising the antigen binding molecule and EOMES-373K-Me in the sample, thereby stratifying the subject into a possible responder to the therapy.

59. The use of claim 54, wherein the method comprises detecting EOMES-641K-Ac in T cells or T cell populations and stratifying patients as likely non-responders to the therapy.

60. The use of claim 59, wherein the method comprises contacting a sample with an antigen binding molecule that specifically binds to EOMES-641K-Ac and detecting a complex comprising the antigen binding molecule and EOMES-641K-Ac in the sample, thereby stratifying patients into potential non-responders to the therapy.

61. The use of claim 54, wherein the method comprises: contacting the sample with a first antigen-binding molecule that specifically binds to EOMES-641K-Ac and a second antigen-binding molecule that specifically binds to PD EOMES-641K-Me; The level of a first complex comprising the first antigen binding molecule and EOMES-641K-Ac and the level of a second complex comprising the second antigen binding molecule and EOMES-641K-Me are measured in the sample; and the subjects are stratified into possible responders or non-responders based on a comparison of the level of the first complex and the level of the second complex, wherein if the level of the first complex is higher than the level of the second complex in the sample, the subject is stratified as a possible non-responder, and wherein if the level of the second complex is higher than the level of the first complex, the subject is stratified as a possible responder.

62. The use of any one of claims 46-52 and 54-61, wherein the method further comprises detecting at least one additional biomarker.

63. The use of claim 62, wherein the at least one additional biomarker is selected from IFN-γ, TNF-α, IL-2, Ki67, PD-1, and CD107a.

64. Use of a reagent for detecting post-translational modifications in the nuclear localization sequence of EOMES and / or the DNA binding motif of EOMES in T cells or T cell populations in the preparation of a preparation for a method for assessing the immune function of a subject, wherein the post-translational modification in the nuclear localization sequence is selected from EOMES-641K-Ac and EOMES-641K-Me, and wherein the post-translational modification in the DNA binding motif is EOMES-373K-Me.

65. The use of claim 64, wherein the method comprises detecting acetylation of EOMES-641K in a T cell or a T cell population, thereby determining that the subject has impaired immune function.

66. The use of claim 65, wherein the method comprises detecting an increased level of EOMES-641K-Ac in a T cell or T cell population relative to a suitable control.

67. The use of claim 64 or 66, wherein the method further comprises detecting the cellular localization of EOMES-641K-Ac in T cells.

68. The use of claim 67, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-641K-Ac in T cells.

69. The use of claim 68, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization or the ratio of cytoplasmic to nuclear localization of EOMES-641K-Ac in T cells.

70. The use of claim 64, wherein the method comprises detecting methylation of EOMES-641K in a T cell or a T cell population, thereby determining that the subject has normal or active immune function.

71. The use of claim 70, wherein the method comprises detecting an increased level of EOMES-641K-Me in a T cell or T cell population relative to a suitable control.

72. The use of claim 70 or 71, wherein the method further comprises detecting the cellular localization of EOMES-641K-Me in T cells.

73. The use of claim 72, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-641K-Me in T cells.

74. The use of claim 73, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization or the ratio of cytoplasmic to nuclear localization of EOMES-641K-Me in T cells.

75. The use of claim 64, wherein the method comprises detecting methylation of EOMES-373K in a T cell or a T cell population, thereby determining that the subject has normal or active immune function.

76. The use of claim 75, wherein the method comprises detecting an increased level of EOMES-373K-Me in a T cell or T cell population relative to a suitable control.

77. The use of claim 75 or 76, wherein the method further comprises detecting the cellular localization of EOMES-373K-Me in T cells.

78. The use of claim 77, wherein the method comprises detecting the nuclear and / or cytoplasmic localization of EOMES-373K-Me in T cells.

79. The use of claim 78, wherein the method comprises detecting the ratio of nuclear to cytoplasmic localization or the ratio of cytoplasmic to nuclear localization of EOMES-373K-Me in T cells.

80. An antigen binding molecule that specifically binds to EOMES-641K-Ac.

81. Use of the antigen binding molecule of claim 80 in the preparation of a preparation for assessing T cell function, predicting the likelihood of a subject with cancer to respond to therapy, determining the likelihood that a subject with cancer is resistant to therapy, determining the likelihood that a subject with cancer is sensitive to therapy, stratifying subjects with cancer into likely responders or non-responders to therapy, managing subjects with cancer with therapy, assessing the immune function of a subject, and / or managing subjects with impaired or decreased immune function with therapy.

82. A complex comprising EOMES-641K-Ac and an antigen-binding molecule that specifically binds to EOMES-641K-Ac.

83. An antigen binding molecule that specifically binds to EOMES-641K-Me.

84. Use of the antigen binding molecule of claim 83 in the preparation of a preparation for assessing T cell function, predicting the likelihood of a subject with cancer to respond to therapy, determining the likelihood that a subject with cancer is resistant to therapy, determining the likelihood that a subject with cancer is sensitive to therapy, stratifying subjects with cancer into likely responders or non-responders to therapy, managing subjects with cancer with therapy, assessing the immune function of a subject, and / or managing subjects with impaired or decreased immune function with therapy.

85. A complex comprising EOMES-641K-Me and an antigen-binding molecule that specifically binds to EOMES-641K-Me.

86. An antigen binding molecule that specifically binds to EOMES-373K-Me.

87. Use of the antigen binding molecule of claim 86 in the preparation of a preparation for assessing T cell function, predicting the likelihood of a subject with cancer to respond to therapy, determining the likelihood that a subject with cancer is resistant to therapy, determining the likelihood that a subject with cancer is sensitive to therapy, stratifying subjects with cancer into likely responders or non-responders to therapy, managing subjects with cancer with therapy, assessing the immune function of a subject, and / or managing subjects with impaired or decreased immune function with therapy.

88. A complex comprising EOMES-373K-Me and an antigen-binding molecule that specifically binds to EOMES-373K-Me.

89. A kit for assessing T cell function, predicting the likelihood of a subject with cancer to respond to therapy, determining the likelihood that a subject with cancer is resistant to therapy, determining the likelihood that a subject with cancer is sensitive to therapy, stratifying subjects with cancer into likely responders or non-responders to therapy, and / or managing subjects with cancer for treatment with therapy, assessing the immune function of a subject, and / or managing subjects with impaired or decreased immune function for treatment with therapy, the kit comprising at least one antigen binding molecule selected from the following: an antigen binding molecule that specifically binds to EOMES-641K-Ac, an antigen binding molecule that specifically binds to EOMES-641K-Me, and an antigen binding molecule that specifically binds to EOMES-373K-Me.

90. The kit of claim 89, further comprising one or more controls, including a positive control and a negative control.

91. The kit of claim 90, wherein the positive control is selected from the group consisting of an EOMES-641K-Ac polypeptide, an EOMES-641K-Me polypeptide, and an EOMES-373K-Me polypeptide.

92. The kit of any one of claims 89-91, further comprising instructional materials for carrying out the use of any one of claims 1-79.

93. An isolated T cell comprising a complex, wherein the complex comprises EOMES-641K-Ac and a first antigen binding molecule that specifically binds to EOMES-641K-Ac; EOMES-641K-Me and a first antigen binding molecule that specifically binds to EOMES-641K-Me; or EOMES-373-Me and a first antigen binding molecule that specifically binds to EOMES-373K-Me.

94. The T cell of claim 93, further comprising a second antigen-binding molecule that binds to the first antigen-binding molecule.

95. The T cell of claim 94, wherein the second antigen binding molecule comprises a detectable label.

96. The use of any one of claims 17-19, 21-24, 26-29, 31-34, 36, 37, 39, 40, 42, 43, 45, 46-52, 54-61, 63, 81, 84, and 87, or the kit of any one of claims 89-91, wherein the therapy is immunotherapy.

97. The use or kit of claim 96, wherein the immunotherapy is an immune checkpoint inhibitor.

98. The use or kit of claim 97, wherein the immune checkpoint inhibitor is an antagonist antigen binding molecule that specifically binds to an immune checkpoint molecule.

99. The use or kit of claim 98, wherein the antagonist antigen binding molecule specifically binds to an immune checkpoint molecule selected from PD-1, PD-L1 and CTLA4.

100. The use of any one of claims 17-19, 21-24, 26-29, 31-34, 36, 37, 39, 40, 42, 43, 45, 46-52, 54-61, 63, 81, 84, and 87, or the kit of any one of claims 89-91, wherein the therapy is a cytotoxic therapy.

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