Patent Application
20210003556
This document relates to materials and methods for identifying cancer patients who are likely to respond to chemo-immunotherapy (CIT), including materials and methods for using CX3CR1 to identify PD-1 therapy-responsive CD8+ T cells that withstand the toxicity of chemotherapy during combined cancer CIT.
Immune checkpoint inhibitor (ICI) therapies targeted to programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) have achieved a durable clinical benefit in a subset of patients with cancer. Unlike chemotherapy or radiation therapy, PD-1 ICI therapy does not directly destroy tumor cells, but rather works through at least two steps: (1) blocking PD-1 signals in T cells; and (2) expanding immune effector cells capable of rejecting tumor cells. However, primary or acquired resistance to PD-1 ICI is common, and is a pressing challenge in cancer immunotherapy. Some cancer patients with tumors that progressed upon anti-PD-1 therapy have benefitted from the addition of salvage chemotherapy, even though cytotoxic chemotherapy has been viewed as toxic to immune cells. The mechanism responsible for the successful clinical outcomes of CIT is not completely understood.
This document is based, at least in part, on the discovery that a subset of tumor-reactive CD8+ T cells, expressing the chemokine receptor CX3CR1, endured cytotoxic chemotherapy and significantly increased in response to combined chemo-immunotherapy (paclitaxel and carboplatin with PD-1 blockade) in metastatic melanoma patients. These CX3CR1+CD8+ T cells have an effector memory phenotype and the ability to efflux chemotherapy drugs via the ABCB1 transporter. This document also is based, at least in part, on the identification of a combination and sequence of CIT that results in an increase in CX3CR1+CD8+ T cells required for mediating tumor regression. The studies described herein define a critical role for CX3CR1+ CD8+ tumor-reactive T cells in the success of CIT, promoting their development as a marker for monitoring patient responses to CIT.
This document also is based, at least in part, on the discovery that % Bim+ CD8+ T cells can be used as a molecular marker for PD-1 blockade-responsiveness. This marker, in combination with the CX3CR1+ CD8+ T cell marker, can be used not only to predict the degree to which PD-1 ICI therapy has turned a patient's immune system to reject tumors, but also to aid in identifying patients who would likely benefit from an appropriate combined therapy. For example, some patients may demonstrate responses to PD-1 blockade (with a decrease of Bim+ CD8+ T cells), but without a clinical response due to lack of sufficient effector cells (CX3CR1+ Granzyme CD8+ T cells). For such patients, continued application of PD-1 ICI may still provide the benefit of preventing CD8+ T cells from apoptosis mediated by high Bim expression, and also provide a window for combined therapy that can reduce tumor burden and expand effector T cells.
In a first aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, where the first population of CD8+ T cells was obtained prior to treatment of the subject with PD-1 blockade therapy; measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a second population of CD8+ T cells obtained from the subject, where the second population of CD8+ T cells was obtained after treatment of the subject with PD-1 blockade therapy; identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by at least a predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is decreased by at least a predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population; and treating the subject with a therapy to increase tumor immunogenicity. The predetermined CX3CR1+ threshold can be an increase of at least 80%, and the predetermined Bim+ threshold can be a decrease of at least 20%. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, time of flight mass cytometry (cyToF), immunohistochemistry (IHC), multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The therapy to increase tumor immunogenicity can include radiation. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations.
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, where the first population of CD8+ T cells was obtained prior to treatment of the subject with PD-1 blockade therapy; measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a second population of CD8+ T cells obtained from the subject, where the second population of CD8+ T cells was obtained after treatment of the subject with PD-1 blockade therapy; identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by less than a predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is decreased by at least a predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population; and treating the subject with cytokine therapy combined with PD-1 blockade therapy. The predetermined CX3CR1+ threshold can be an increase of at least 80%, and the predetermined Bim+ threshold can be a decrease of at least 20%. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The cytokine therapy can include treatment with IL-15. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations.
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, where the first population of CD8+ T cells was obtained prior to treatment of the subject with PD-1 blockade therapy; measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a second population of CD8+ T cells obtained from the subject, where the second population of CD8+ T cells was obtained after treatment of the subject with PD-1 blockade therapy; identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by at least a predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is increased, is unchanged, or is decreased by less than a predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population; and treating the subject with combined CIT. The predetermined CX3CR1+ threshold can be an increase of at least 80%, and the predetermined Bim+ threshold can be a decrease of at least 20%. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The CIT can include treatment with paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations.
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, where the first population of CD8+ T cells was obtained prior to treatment of the subject with PD-1 blockade therapy; measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a second population of CD8+ T cells obtained from the subject, where the second population of CD8+ T cells was obtained after treatment of the subject with PD-1 blockade therapy; identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by less than a predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is increased, is unchanged, or is decreased by less than a predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population; and treating the subject with an ICI therapy other than PD-1 blockade, optionally in combination with chemotherapy. The predetermined CX3CR1+ threshold can be an increase of at least 80%, and the predetermined Bim+ threshold can be a decrease of at least 20%. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The ICI therapy can include treatment with anti-TIGIT and/or anti-Tim 3. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations.
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells within a population of CD8+ T cells obtained from a subject having a tumor, identifying the subject as being likely to respond to combined CIT when the percentage of CX3CR1+ cells within the population is increased relative to a corresponding control percentage of CX3CR1+ cells, and administering the CIT to the subject. The population of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The method can include obtaining the population of CD8+ T cells before treatment of the subject with the CIT, after treatment of the subject with the CIT, after treatment of the subject with chemotherapy (e.g., paclitaxel, carboplatin, or a combination thereof) or after treatment of the subject with ICI therapy (anti-PD-1 or anti-PD-L1 therapy). The CIT can include paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The corresponding control percentage can be the percentage of CX3CR1+ cells in a population of CD8+ T cells obtained from the subject at baseline. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the population, and identifying the subject as being likely to respond to the CIT when the percentage of CX3CR1+ Granzyme B+ cells within the population is increased relative to a corresponding control percentage of CX3CR1+ Granzyme B+ cells (e.g., the percentage of CX3CR1+ Granzyme B+ cells in a population of CD8+ T cells obtained from the subject at baseline). The method can further include measuring the percentage of Bim+ CD8+ T cells within the population, and identifying the subject as being likely to respond to CIT when the percentage of Bim+ CD8+ T cells within the population is decreased relative to a corresponding control percentage of Bim+ CD8+ T cells (e.g., the percentage of Bim+ cells in a population of CD8+ T cells obtained from the subject at baseline).
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, wherein the first population was obtained from the tumor prior to CIT, administering the CIT to the subject, measuring the percentage of CX3CR1+ cells within a second population of CD8+ T cells obtained from the subject, wherein the second population was obtained from the tumor after CIT, and identifying the subject as being responsive to the CIT when the percentage of CX3CR1+ cells within the second population is increased relative to the percentage of CX3CR1+ cells within the first population. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The method can include obtaining the first population of CD8+ T cells after treatment of the subject with chemotherapy (e.g., paclitaxel, carboplatin, or a combination thereof), or after treatment of the subject with ICI therapy (e.g., anti-PD-1 or anti-PD-L1 therapy). The CIT can include paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations, and identifying the subject as being responsive to the CIT when the percentage of CX3CR1+ Granzyme B+ cells within the second population is increased relative the percentage of CX3CR1+ Granzyme B+ cells within the first population. The method can further include measuring the percentage of Bim+ CD8+ T cells within the first and second populations, and identifying the subject as being responsive to the CIT when the percentage of Bim+ CD8+ T cells within the second population is decreased relative to the percentage of Bim+ CD8+ T cells within the first population.
In yet another aspect, this document features a method that includes obtaining a population of CD8+ T cells from a subject having a tumor, measuring the percentage of CX3CR1+ Granzyme B+ cells within the population of CD8+ T cells, identifying the subject as being likely to respond to CIT when the percentage of CX3CR1+ Granzyme B+ cells within the population is increased relative to a corresponding control percentage; and administering the CIT to the subject. The population of CD8+ T cells can be obtained from the peripheral blood of the subject, or from the tumor. The method can include obtaining the population of CD8+ T cells before treatment of the subject with the CIT, after treatment of the subject with the CIT, after treatment of the subject with chemotherapy (e.g., paclitaxel, carboplatin, or a combination thereof), or after treatment of the subject with ICI therapy (e.g., anti-PD-1 or anti-PD-L1 therapy). The CIT can include paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The corresponding control percentage can be the percentage of CX3CR1+ Granzyme B+ cells in a population of CD8+ T cells obtained from the subject at baseline. The method can further include measuring the percentage of Bim+ CD8+ T cells within the population, and identifying the subject as being likely to respond to CIT when the percentage of Bim+ CD8+ T cells within the population is decreased relative to a corresponding control percentage of Bim+ CD8+ T cells (e.g., the percentage of Bim+ cells in a population of CD8+ T cells obtained from the subject at baseline).
In another aspect, this document features a method that includes measuring the percentage of CX3CR1+ Granzyme B+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, wherein the first population was obtained from the tumor prior to CIT, administering the CIT to the subject, measuring the percentage of CX3CR1+ Granzyme B+ cells within a second population of CD8+ T cells obtained from the subject, wherein the second population was obtained from the tumor after CIT, and identifying the subject as being responsive to the CIT when the percentage of CX3CR1+ Granzyme B+ cells within the second population is increased relative to the percentage of CX3CR1+ Granzyme B+ cells within the first population. The first and second populations of CD8+ T cells can be obtained from the peripheral blood of the subject or from the tumor. The method can include obtaining the first population of CD8+ T cells after treatment of the subject with chemotherapy (e.g., paclitaxel, carboplatin, or a combination thereof), or after treatment of the subject with ICI therapy (e.g., anti-PD-1 or anti-PD-L1 therapy). The CIT can include paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis. The method can include measuring the percentage of CX3CR1+ Granzyme B+ cells within the first and second populations, and identifying the subject as being responsive to the CIT when the percentage of CX3CR1+ Granzyme B+ cells within the second population is increased relative the percentage of CX3CR1+ Granzyme cells within the first population. The method can further include measuring the percentage of Bim+ CD8+ T cells within the first and second populations, and identifying the subject as being responsive to the CIT when the percentage of Bim+ CD8+ T cells within the second population is decreased relative to the percentage of Bim+ CD8+ T cells within the first population.
This document also features a method for expanding a population of CX3CR1+ CD8+ T cells, where the method includes obtaining a population of CX3CR1+ CD8+ T cells from a subject, contacting the population with interleukin-15 (IL-15), and determining that the population of CX3CR1+ CD8+ T cells has expanded. The population of CD8+ T cells can be obtained from the peripheral blood of the subject, or from a tumor in the subject. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The determining can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis to assess the number of CX3CR1+ CD8+ T cells. The method can further include administering at least a portion of the expanded CX3CR1+ CD8+ T cell population to the subject.
In addition, this document features a method that includes measuring the percentage of CX3CR1+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, administering IL-15 to the subject, measuring the percentage of CX3CR1+ cells within a second population of CD8+ T cells obtained from the subject after the IL-15 administration, and determining that the percentage of CX3CR1+ cells within the second population is increased relative to the percentage in the first population. The first and second populations of CD8+ T cells can be within a peripheral blood sample from the subject, or from a tumor within the subject. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis to assess the number of CX3CR1+ CD8+ T cells.
In still another aspect, this document features a method for identifying a subject in need of treatment modification. The method can include measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a first population of CD8+ T cells obtained from a subject having a tumor, wherein the first population of CD8+ T cells was obtained prior to treatment of the subject with PD-1 blockade therapy; measuring the percentage of CX3CR1+ cells and the percentage of Bim+ cells within a second population of CD8+ T cells obtained from the subject, wherein the second population of CD8+ T cells was obtained after treatment of the subject with PD-1 blockade therapy; identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by at least a predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population, or is increased by less than the predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population; and identifying the subject as having a percentage of Bim+ cells within the second population that is decreased by at least a predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, or is increased, unchanged, or decreased by less than the predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, thereby identifying the subject as being in need of a therapy other than or in addition to the PD-1 blockade therapy. The method can include identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by at least the predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is decreased by at least the predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, thereby identifying the subject as being in need of a therapy to increase tumor immunogenicity (e.g., a therapy that includes radiation). The method can include identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by less than the predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is decreased by at least the predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, thereby identifying the subject as being in need of cytokine therapy (e.g., treatment with IL-15) combined with PD-1 blockade therapy. The method can include identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by at least the predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is increased, is unchanged, or is decreased by less than the predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, thereby identifying the subject as being in need of CIT (e.g., treatment with paclitaxel, carboplatin, and anti-PD-1 or anti-PD-L1 therapy). The method can include identifying the subject as having a percentage of CX3CR1+ cells within the second population that is increased by less than the predetermined CX3CR1+ threshold relative to the percentage of CX3CR1+ cells within the first population and as having a percentage of Bim+ cells within the second population that is increased, is unchanged, or is decreased by less than the predetermined Bim+ threshold relative to the percentage of Bim+ cells within the first population, thereby identifying the subject as being in need of an ICI therapy other than PD-1 blockade (e.g., treatment with anti-TIGIT and/or anti-Tim 3), optionally in combination with chemotherapy. The predetermined CX3CR1+ threshold can be an increase of at least 80%. The predetermined Bim+ threshold can be a decrease of at least 20%. The predetermined CX3CR1+ threshold can be an increase of at least 80% and the predetermined Bim+ threshold can be a decrease of at least 20%. The first and second populations of CD8+ T cells can be from the peripheral blood of the subject, or can be from the tumor. The subject can be a human. The tumor can contain metastatic melanoma cells, gastrointestinal cancer cells, genitourinary cancer cells, non-small lung cancer cells, or breast cancer cells. The measuring can include using flow cytometry, cyToF, IHC, multiplex immunofluorescence imaging analysis, or single cell or sorted cell-RNA-sequencing analysis.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides materials and methods for identifying patients as being likely to respond to combined CIT, as well as materials and methods for determining optimal therapies and therapeutic timing, and methods and materials for treating cancer. For example, this document provides methods and materials for identifying a subject (e.g., a mammal such as a human) as having an increase in the percentage of CD8+ T cells that are CX3CR1+ (also referred to % CX3CR1+ CD8+ T cells), where the cells are from, e.g., a tumor or the peripheral blood, and classifying that subject as likely to be responsive to treatment with a combination of immunotherapy (e.g., ICI) and chemotherapy (known as CIT). The increase can be relative to a corresponding control percentage, or relative to a previously established percentage for the subject being assessed. In some cases, the methods also can include treating the identified subject with CIT. As described herein, an increased % CX3CR1+ CD8+ T cells can be related to increased efflux of chemotherapy drugs, as well as increased effector memory phenotype.
Having the ability to identify mammals as having a tumor that is likely to respond to a certain treatment (e.g., CIT, ICI, or a combination of CIT and ICI) can allow those mammals to be properly identified and treated in an effective and reliable manner. For example, the disease treatments described herein (e.g., CIT, ICI, and a combination of CIT and ICI) can be used to treat cancer patients identified as having a tumor that is identified as likely to respond to such treatment.
The methods provided herein, in some embodiments, can include identifying a subject as having an increased % CX3CR1+ Granzyme B+ CD8+ T cells, increased % CX3CR1+ CD8+ T cells in combination with decreased % Bim+ CD8+ T cells, or increased % CX3CR1+ Granzyme B+ CD8+ T cells in combination with decreased % Bim+ CD8+ T cells, relative to a corresponding control or previously established percentage for that subject. Subjects who are identified according to any of these criteria can be classified as being likely to respond to CIT. Conversely, subjects who are identified as not having an increased % CX3CR1+ CD8+ T cells, increased % CX3CR1+ Granzyme B+ CD8+ T cells, increased % CX3CR1+ CD8+ T cells in combination with decreased % Bim+ CD8+ T cells, or increased % CX3CR1+ Granzyme B+ CD8+ T cells in combination with decreased % Bim+ CD8+ T cells, relative to a corresponding control or previously established percentage for that subject, can be classified as not being as likely to respond to CIT.
The term “increased” as used herein with respect to % CX3CR1+ CD8+ T cells or % CX3CR1+ Granzyme B+ CD8+ T cells refers to a percentage that is greater (e.g., at least 5% greater, at least 10% greater, at least 25% greater, at least 50% greater, 5 to 10% greater, 10 to 25% greater, 25 to 50% greater, 50 to 75% greater, at least 2-fold greater, at least 3-fold greater, at least 5-fold greater, 2- to 3-fold greater, or 3- to 5-fold greater) than a reference % CX3CR1+ CD8+ T cells or % CX3CR1+ Granzyme B+ CD8+ T cells. The term “decreased” as used herein with respect to % Bim+ CD8+ T cells refers to a percentage that is less (e.g., at least 5% less, at least 10% less, at least 25% less, at least 50% less, at least 75% less, at least 90% less, at least 95% less, 5 to 10% less, 10 to 25% less, 25 to 50% less, 50 to 75% less, or 75 to 100% less) than a reference % Bim+ CD8+ T cells.
The terms “reference %,” “reference percentage” and “reference level” (also referred to herein as “corresponding control %,” “corresponding control percentage,” and “corresponding control level”), as used herein with respect to CX3CR1+ CD8+ T cells, CX3CR1+ Granzyme B+ CD8+ T cells, and Bim+ CD8+ T cells, refer to the % CX3CR1+ cells, % CX3CR1+ Granzyme B cells, or % Bim+ cells in a sample of CD8+ T cells taken from a subject at baseline (e.g., prior to treatment with ICI or chemotherapy).
The presence of an increased % CX3CR1+ CD8+ T cells, increased % CX3CR1+ Granzyme B+ CD8+ T cells, or decreased % Bim+ CD8+ T cells can be determined using, for example, flow cytometry according to the methods described in the Examples herein. In some cases, methods such as time of flight mass cytometry (cyToF), single cell or sorted cell-RNA-sequencing analysis cell staining, western blotting, multiplex immunofluorescence imaging analysis, immunohistochemistry (IHC), or other immunological techniques can be used.
The populations of CD8+ T cells used in the methods provided herein can be from any suitable source within the subject. In some cases, for example, the CD8+ T cells are obtained from the peripheral blood of the subject, while in other cases, the CD8+ T cells are from a tumor within the subject. Other suitable sources include, for example, ascite samples and lymphoid organ samples.
Thus, in some embodiments, this document provides methods that include measuring the % CX3CR1+ cells within a population of CD8+ T cells obtained from a subject that has a tumor, and identifying the subject as being likely to respond to CIT when the % CX3CR1+ cells within the population is increased relative to a corresponding control % CX3CR1+ cells. The methods also can include measuring the % CX3CR1+ Granzyme B+ cells within the population of CD8+ T cells from the subject; in such embodiments, the subject can be identified as likely to respond to CIT when the % CX3CR1+ Granzyme B+ cells within the population is increased relative to a corresponding control percentage. In some cases, the methods also may include administering the CIT to the subject.
This document also provides methods that can include measuring the % CX3CR1+ cells in a first population of CD8+ T cells obtained from a subject with a tumor prior to CIT, measuring the % CX3CR1+ cells in a second population of CD8+ T cells obtained from the subject after CIT, and identifying the subject as being responsive to the CIT when the % CX3CR1+ cells in the second population is greater than the % CX3CR1+ cells in the first population. In some cases, the methods can include measuring the % CX3CR1+ Granzyme B+ cells in the first and second populations of CD8+ T cells, and identifying the subject as being responsive to the CIT when the % CX3CR1+ Granzyme B+ cells in the second population is greater than the % CX3CR1+ Granzyme B+ cells in the first population. In some cases, the methods also can include administering the CIT to the subject.
As described herein, the percentage of Bim+ cells in a population of CD8+ T cells can be inversely correlated with the percentage of CX3CR1+ or CX3CR1+ Granzyme B+ cells in the population. In some cases, therefore, the methods provided herein also can utilize the % Bim+ CD8+ T cells as an indicator that a subject is likely to respond to CIT or another therapy. Such methods can include, for example, measuring the % Bim+ CD8+ T cells within a population of CD8+ T cells evaluated for CX3CR1, or CX3CR1 and Granzyme B, and identifying the subject as being likely to respond to CIT when the % Bim+ CD8+ T cells within the population is decreased relative to a corresponding control % Bim+ CD8+ T cells.
In some cases, the change in % CX3CR1+ CD8+ T cells (or % CX3CR1+ Granzyme B+ T cells) and the change in % Bim+ CD8+ T cells from a reference percentage in a sample from a subject (e.g., before treatment of the subject with ICI, CIT, or chemotherapy) can be used to determine a therapy that is likely to benefit the subject. Samples containing CD8+ T cells obtained from the subject before and after treatment (e.g., with an ICI therapy such as anti-PD-1 therapy) can be assessed to determine the % CX3CR1+ and % Bim+ CD8+ T cells in the samples, and a further treatment regimen can be determined based, at least in part, on whether the changes in % CX3CR1+ CD8+ T cells and Bim+ CD8+ T cells reach certain predetermined thresholds.
For example, when the % CX3CR1+ cells in the second population is increased by at least a predetermined threshold relative to the % CX3CR1+ cells within the first population, and the % Bim+ cells in the second population is decreased by at least a predetermined threshold relative to the % Bim+ cells in the first population, it may be determined that they subject is likely to benefit from a therapy that can increase tumor immunogenicity (e.g., radiation therapy). When the % CX3CR1+ cells in the second population is increased by less than the predetermined CX3CR1+ threshold and the % Bim+ cells in the second population is decreased by at least the predetermined Bim+ threshold, it may be determined that the subject is likely to benefit from cytokine therapy (e.g., treatment with IL-15) combined with PD-1 blockade therapy. When the % CX3CR1+ cells in the second population is increased by at least the predetermined CX3CR1+ threshold and the % Bim+ cells in the second population is increased, unchanged, or decreased by less than the predetermined Bim+ threshold, it may be determined that the subject is likely to benefit from CIT. When the % CX3CR1+ cells in the second population is increased by less than the predetermined CX3CR1+ threshold and the % Bim+ cells in the second population is increased, unchanged, or decreased by less than the predetermined Bim+ threshold, it may be determined that the subject is likely to benefit from an ICI therapy other than PD-1 blockade therapy (e.g., anti-TIGIT (T cell immunoreceptor with Ig and ITIM domains) therapy and/or anti-Tim 3 therapy), optionally in combination with chemotherapy.
The predetermined thresholds can be established using methods such as those described in the examples herein. In some embodiments, a predetermined CX3CR1 threshold can be an increase of at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%), and a predetermined Bim threshold can be a decrease of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%).
The populations of CD8 T cells used in the methods described herein can be obtained from a subject at any suitable time. For example, CD8+ T cells can be obtained before or after (e.g., six, 12, 16, 32, two to four, four to six, six to eight, eight to 12, 12 to 16, 16 to 32, or more than 32 weeks after) treatment with CIT, before or after treatment with chemotherapy (e.g., paclitaxel and/or carboplatin), or before or after ICI therapy (e.g., with an anti-PD-1 or anti-PD-L1 antibody), or when disease progresses.
The subject can be a mammal (e.g., a human, non-human primate, mouse, rat, rabbit, pig, sheep, dog, cat, or horse), and can have a tumor such as, without limitation, a melanoma (e.g., a metastatic melanoma), a gastrointestinal tumor, a genitourinary tumor, a non-small cell lung cancer, or a breast tumor.
In addition, this document provides methods that can be used to expand CX3CR1+ CD8 T cells, either in vitro, ex vivo, or in vivo. Such methods can utilize interleukin-15 (IL-15) to stimulate expansion of the cells, as described in Example 8 herein; methods also can utilize IL-12, IL-2 and IL-7, and/or fractalkine (a CX3CR1 ligand) to stimulate expansion of the cells. Thus, in some embodiments, the methods provided herein can include obtaining a population of CX3CR1+ CD8+ T cells from a subject and then contacting the population with IL-15 in order to expand the population. In some cases, the methods can further include returning at least a portion of the expanded population to the subject from which they were obtained (e.g., to combat a tumor, for example). Methods for in vivo use can include, for example, measuring the % CX3CR1+ cells in a first population of CD8+ T cells obtained from a subject with a tumor, administering IL-15 to the subject, measuring the % CX3CR1+ cells in a second population of CD8+ T cells obtained from the subject after IL-15 administration to demonstrate that the % CX3CR1+ cells within the second population has increased relative to the % CX3CR1+ cells in the first population.
In some embodiments, once a subject has been identified as having an increased % CX3CR1+ CD8+ T cells, increased % CX3CR1+ Granzyme B+ CD8+ T cells, or increased % CX3CR1+ or % CX3CR1+ Granzyme B+ CD8+ T cells in combination with decreased % Bim+ CD8+ T cells, the subject can be treated with one or more cancer therapies. Examples of such therapies include, without limitation, chemotherapies such as paclitaxel, carboplatin, cisplatin, doxorubicin, or gemcitabine, ICI therapies targeted to PD-1 or PD-L1, a combination of ICI therapy and chemotherapy (CIT), and radiation. Methods for administering such therapies are known in the art. Administration can be, for example, parenteral (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). In some embodiments, administration can be topical (e.g., transdermal, sublingual, ophthalmic, or intranasal), pulmonary (e.g., by inhalation or insufflation of powders or aerosols), or oral. In addition, a therapy can be administered prior to, after, or in lieu of surgical resection of a tumor.
A cancer therapy (e.g., chemotherapy or immunotherapy, or a CIT) can be administered to a mammal in an appropriate amount, at an appropriate frequency, and for an appropriate duration effective to achieve a desired outcome (e.g., to increase progression-free survival, reduce tumor size, etc.). In some cases, a therapy can be administered to a subject having cancer to reduce the progression rate of the cancer by at least 5 percent (e.g., at least 5 percent, at least 10 percent, at least 25 percent, at least 50 percent, at least 75 percent, or 100 percent). For example, the progression rate can be reduced such that no additional cancer progression is detected. Any appropriate method can be used to determine whether or not the progression rate of cancer is reduced. For skin cancer (e.g., melanoma), for example, the progression rate can be assessed by imaging tissue at different time points and determining the amount of cancer cells present. The amounts of cancer cells measured in tissue at different times can be compared to determine the progression rate. After treatment, the progression rate can be determined again over another time interval. In some cases, the stage of cancer after treatment can be determined and compared to the stage before treatment to determine whether or not the progression rate has been reduced.
In some cases, a therapy can be administered to a subject having cancer under conditions where progression-free survival is increased (e.g., by at least 5, at least 10, at least 25, at least 50, at least 75, or at least 100 percent) as compared to the median progression-free survival of corresponding subjects having untreated cancer, or the median progression-free survival of corresponding subjects having cancer and treated with other therapies. Progression-free survival can be measured over any length of time (e.g., one month, two months, three months, four months, five months, six months, or longer).
An effective amount of a composition containing a molecule as provided herein can be any amount that reduces tumor size, reduces the progression rate of cancer, increases the progression-free survival rate, or increases the median time to progression without producing significant toxicity to the mammal. Optimum dosages can vary depending on the relative potency of individual therapies (e.g., antibodies and chemotherapeutics), and can generally be estimated based on EC50 found to be effective in in vitro and in vivo animal models. Typically, dosage is from 0.01 μg to 100 g per kg of body weight. For example, an effective amount of an antibody or fusion protein can be from about 1 mg/kg to about 100 mg/kg (e.g., about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 50 mg/kg, about 75 mg/kg, about 5 to 10 mg/kg, about 10 to 20 mg/kg, about 20 to 50 mg/kg, or about 75 to 100 mg/kg). If a particular subject fails to respond to a particular amount, then the amount of the therapy can be increased by, for example, two-fold. After receiving this higher concentration, the subject can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the cancer may require an increase or decrease in the actual effective amount administered.
The frequency of administration can be any frequency that reduces tumor size, reduces the progression rate of cancer, increases the progression-free survival rate, or increases the median time to progression without producing significant toxicity to the subject. For example, the frequency of administration can be once or more daily, biweekly, weekly, monthly, or even less. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment can include rest periods. For example, a composition containing an immunotherapy can be administered over a two week period followed by a two week rest period, and then repeated or followed by treatment with chemotherapy. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the cancer may require an increase or decrease in administration frequency.
An effective duration for administering a therapy can be any duration that reduces tumor size, reduces the progression rate of cancer, increases the progression-free survival rate, or increases the median time to progression without producing significant toxicity to the subject. Thus, the effective duration can vary from several days to several weeks, months, or years. In general, the effective duration for the treatment of cancer can range in duration from several weeks to several months. In some cases, an effective duration can be for as long as an individual subject is alive.
Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the cancer.
After administering a therapy to a subject with cancer, the subject can be monitored to determine whether or not the cancer was treated. For example, a subject can be assessed after treatment to determine whether or not the progression rate of the cancer has been reduced (e.g., stopped), or whether the tumor size has decreased. Any method, including those that are standard in the art, can be used to assess progression and survival rates.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Patient information: The studies described herein were conducted according to Declaration of Helsinki principles. Peripheral blood and tissue samples for this study were collected after written consents were obtained. Clinical course, treatment information and outcomes in patients with metastatic melanoma who did not respond to anti-PD-1 (programmed cell death protein 1) single agent therapy were retrospectively collected. Patients who failed initial PD-1 therapy were subsequently treated with salvage paclitaxel and carboplatin combination in addition to PD-1 blockade, regardless of BRAF mutant status. Response to treatment was evaluated according to standard clinical practice guidelines using Response Evaluation Criteria In Solid Tumors (RECIST) criteria.
Flow analysis of human T cells isolated from peripheral blood: PBMC samples were collected from healthy donors or patients with melanoma. Antibodies for CD45, CD3, CD8, CX3CR1 (2A9-1), CD11a (HI111) and PD-1 (EH12.2H7) were purchased from BioLegend (San Diego, Calif.); anti-human Granzyme B (GB11) was purchased from Life Technologies (Waltham, Mass.). CD8+ T cells were first stained for surface markers (CX3CR1, etc.), followed by intracellular staining for Granzyme B. To initiate CTL (cytotoxic T lymphocyte) function, cells were briefly stimulated with phosphomolybdic acid (PMA) and ionomycin (Sigma; St. Louis, Mo.) for 5 hours in the presence of anti-CD107a antibody (H4A3), followed by intracellular staining of anti-IFN-γ antibody (4S.B3). Flow cytometry analysis was performed using FlowJo software (Tree Star; Ashland, Oreg.).
RNA-seq and bioinformatics analysis: Total RNA was extracted from flow sorted cells using an RNeasy Mini kit (Qiagen; Hilden, Germany) and checked for quality by Bioanalyzer (RNA 6000 Pico kit; Agilent; Santa Clara, Calif.). A total of 1 ng of RNA was used to generate double stranded cDNA using SMARTER™ Ultra Low RNA kit for Illumina (Takara; Mountain View, Calif.). Full length, double stranded cDNA was quantified and subjected to RNA-Seq library construction. A total of 250 pg of cDNAs were used to construct indexed libraries using NEXTERA® XT DNA Sample Preparation kit (Illumina; San Diego, Calif.). The cDNA and NGS libraries were quantified using Bioanalyzer (High Sensitivity DNA analysis kit; Agilent) and Qubit (dsDNA BR Assay kits; Life Technologies). The libraries were sequenced using the 101 bases paired-end protocol on Illumina HiSeq 2000. FASTQ formatted raw files from each sample were mapped and aligned to reference hg19.
The MAPRSeq workflow for mRNA was used to align raw FASTQ reads, using TopHat2 to the relevant genome. The BAM files thus obtained were passed through other tools for further analysis. Fusion detection was done using a module from the TopHat aligner, called TopHat-Fusion. Raw and normalized gene and exon counts were generated by FeatureCounts, which uses the ENSEMBL GRCh38.78 gene definitions. An in-house tool (RVBoost; Wang et al., Bioinformatics, 2014, 30(23):3414-3416), which uses UnifiedGenotyper from GATK, was employed to report single nucleotide variants present in the data. Finally, the RSeQC module created a variety of QC plots and graphs to ensure that the quality of samples was good and reliable for use in further downstream analyses (e.g., differential expression and pathway analysis). The R-based tool from Bioconductor, edgeR v3.8.6, was used to perform the differential expression analysis comparing the various sample groups. Genes encoded by mRNA that had an absolute log 2 fold change >1.5 were considered to be significantly differently expressed. Heatmaps were created using the heatmap.2 function of the gplots package from R.
Immunochemistry staining of melanoma tissues: Paraffin-embedded tissue sections were cut into 5 m sections, deparaffinized in xylene, and rehydrated in a graded series of alcohols. Antigen retrieval was performed by heating tissue sections in Target Retrieval Solution pH 6.0 (Dako #S1699) at 98° C. for 30 minutes. Sections were cooled on the bench for 20 minutes, washed in running DH20 for 5 minutes, and then incubated for 5 minutes in wash buffer. Sections were then blocked for 5 minutes with Endogenous Peroxidase Block (Dako #S2001), washed, and blocked for 5 minutes in Protein Block Serum Free (Biocare Medical #X0909). Slides were incubated for one hour in mouse monoclonal anti-human Granzyme B (Dako #M7235) diluted 1:50 in Antibody Diluent with Background Reducers (Dako #3022). Sections were washed and incubated 15 minutes each in mouse probe and mouse polymer AP (Mach 3 Mouse AP Polymer Detection Kit, Biocare Medical #M3M532L). Sections were incubated for 5 minutes in Warp Red Chromogen (Biocare Medical #WR806H) for visualization. Subsequently, sections were incubated for 5 minutes in 80° C. Citrate Buffer pH 6, rinsed in wash buffer and incubated in Protein Block Serum Free for 5 minutes. Rabbit anti-human CX3CR1 (Invitrogen PA5-32713) was applied to sections at 1:500 dilution and incubated for one hour at room temperature. Sections were washed and incubated for 15 minutes each in rabbit probe and rabbit polymer HRP (Mach 3 Rabbit HRP Polymer Detection kit, Biocare Medical # M3R531L) and visualized for one minute in DAB (Biocare Medical #BDB2004L). Sections were counterstained and coverglass mounted with PERMOUNT™.
Stimulation and culture of human T cells: Human CD8+ T cells were purified using a human CD8+ T cell enrichment kit (Stemcell). CD8+ T cells were incubated with chemotherapy drugs (paclitaxel, carboplatin, or doxorubicin), either alone or with T cell activators (DYNABEADS®, human T-activator CD3/CD28 beads) for 24-48 hours, followed with staining for CX3CR1 and Granzyme B. ABCB1 inhibitor PGP4008 was purchased from Enzo Life Sciences (Farmingdale, N.Y.).
Drug efflux assay in T cells: Human primary CD8+ T cells were isolated from peripheral blood and incubated (loading) with Rh123 (10 μg/ml) on ice for 30 minutes, or with doxorubicin (Dox, 1 μg/ml) at 37° C. for 60 minutes in water bath. After the loading process, cells were washed and cultured at 37° C. for 60 minutes (efflux), stained for cell surface markers, and analyzed by flow cytometry. The ABCB1 inhibitor PGP-4008 was added at 1-5 μM during the efflux process.
Animal models for chemo-immunotherapy: Both wild type and CX3CR1-deficient (KO) mice in the C57BL/6 background were purchased from Jackson Lab (Bar Harbor, Me.) and maintained under pathogen-free conditions. B16F10 mouse melanoma cells (1×105) were subcutaneously (s.c.) injected into mice in the right flank, followed by i.p. injection of 100 g anti-PD-1 (G4), anti-PD-L1 (10B5), or control IgG starting on day 7, for a total of five doses at 3-day intervals. Carboplatin (40 μg/g plus paclitaxel (10 μg/g body weight) were injected i.p. once, either on day 7 or on day 10 after tumor injection. CTL function of tumor-infiltrating CD8+ T cells was measured by briefly stimulating them with PMA and ionomycin (Sigma) for 5 hours in the presence of anti-CD107a antibody (1D4B), followed by intracellular staining with anti-IFN-γ antibody (XMG1.2). Perpendicular tumor diameters were measured using a digital caliper and tumor sizes were calculated as length×width. Tumor growth was evaluated every 2 to 3 days until ethical endpoints, when all mice were euthanized.
T cell transfer therapy: Spleen cells isolated from OT-1 mice expressing OVA-antigen-specific TCR were cultured with OVA peptide (1 μg/ml) and rhIL-2 (10 IU/ml) for 48 hours. CX3CR1+ and CX3CR1− CD8+ T cells were sorted after culture on the day of T cell transfer. Once B16-OVA mouse melanoma tumors were established, around day 7 after tumor cell injection (5×105 cells per mouse, s.c.), the animals were treated by i.t. injection of CX3CR1+ or CX3CR1− CD8+ T cells at equal numbers (2 to 3×105 T cells per mouse) for a total of three doses on days 7, 10, and 13 after tumor injection.
Statistics: The Mann-Whitney test was used to compare independent groups (function or subsets of CD8+ T cells). The impact of chemotherapy and anti-PD antibody on tumor growth were analyzed by two-way ANOVA. Comparisons of the impact of ABCB1 inhibitors on the efflux of drug were analyzed with one-way ANOVA due to the numerical independent variables. The survival of animals was analyzed by Log-rank Mantel-Cox test. All statistical analyses were performed using GraphPad Prism software 5.0 (GraphPad Software, Inc.; San Diego, Calif.). A P value <0.05 was considered statistically significant.
A large fraction of cancer patients (60-70%) who receive PD-1 blockade alone are resistant to PD-1 therapy or experience subsequent disease progression (Robert et al., N Engl J Med, 2015, 372(26):2521-2532; Robert et al., N Engl J Med, 2015, 372(4):320-330; and Ribas et al., JAMA, 2016, 315(15):1600-1609). Some of these patients, however, benefited from late-line or salvage treatment with conventional chemotherapy. Since the safety and efficacy profile of CIT have been demonstrated in NSCLC patients (Rizvi et al., J Clin Oncol, 2016, 34(25):2969-2979; and Langer et al., Lancet Oncol, 2016, 17(11):1497-1508), a number of patients who had evidence of disease progression with initial PD-1 blockade monotherapy were empirically treated with chemotherapy in addition to continued anti-PD-1 antibody (Yan et al., “The Mayo Clinic experience in patients with metastatic melanoma who have failed previous pembrolizumab treatment,” ASCO Meeting Abstracts. 2016, 34((15_suppl)):e21014). To minimize toxicity, a short-term chemotherapy (2-6 cycles) was combined with anti-PD-1 therapy that was maintained thereafter. Among 19 patients who did not respond to anti-PD-1 (Pembrolizumab) antibody and received chemotherapy (carboplatin, paclitaxel, temozoromed, or dacarbazine,), a complete follow up identified 5 patients demonstrating disease control, with an objective response rate (ORR) of 26.3% according to the RECIST criteria (Seymour et al., Lancet Oncol, 2017, 18(3):e143-e152).
Studies were conducted to seek biomarkers for identifying responders to anti-PD-1 therapy, in order to predict and increase the efficacy of chemo-immunotherapy. First, subsets of tumor-reactive CD8+ T cells were examined in the peripheral blood of cancer patients to identify those that would be responsive to anti-PD-1 monotherapy. Further studies were directed at determining whether the responsive T cell population would be preserved during chemotherapy and would still be responsive to anti-PD-1 therapy. To that end, RNA-seq analysis was performed with of tumor-reactive CD11ahighPD-1+ CD8+ T cells (Liu et al., Oncoimmunology, 2013, 2(6):e23972), and gene transcription was compared between responders and non-responders at baseline prior to PD-1 therapy. Among the top genes with increased expression (ratio >1.5) in responders compared to non-responders, transcription of CX3CR1 was increased in the tumor-reactive CD8+ T cells in the peripheral blood of responders to PD-1 therapy (
The gene expression in CD11ahigh CD8+ T cells isolated and sorted from the peripheral blood of 3 months after anti-PD-1 treatment was then compared between responders and non-responders. As shown in
To further confirm whether CX3CR1 can identify PD-1 therapy-responsive CD8+ T cells, the expression of PD-1 was measured and compared among CX3CR1+ or CX3CR1− CD8+ T cells. As shown in
The frequency of CX3CR1+ Granzyme B+ CD8+ tumor-reactive T cells was examined before and after chemotherapy combined with anti-PD-1 therapy in patients with metastatic melanoma. As shown in
The mechanisms by which CX3CR1+ CD8+ T cells withstand chemotherapy were examined. High multidrug efflux capacity can confer upon CD8+ T cells the ability to survive cytotoxic chemotherapy (Turtle et al., Immunity, 2009, 31(5):834-844). To determine whether high efflux capacity contributes to the survival of CX3CR1+ CD8+ T cells during chemotherapy, the efflux of a fluorescent anthracycline (doxorubicin) was measured in human primary CD8+ T cells isolated from healthy donors. The efflux of doxorubicin increased overtime in CX3CR1 CD8+ T cells (
Since the pharmacodynamics of doxorubicin may not be able to exactly reflect the efflux of carboplatin and paclitaxel (CP), and these drugs cannot be directly tracked due to lack of fluorescent capability, the impact of the drug transporter inhibitor on the function of T cells in the presence of CP was examined to determine whether T cell function might be dampened due to the reduced ability of T cells to efflux CP. ABCB1 transporter inhibitor (PGP4008) was incubated with resting or activated human primary CD8+ T cells in vitro in the presence of CP. T cell function was measured by based pm degranulation (CD107a expression) and intracellular IFN-γ production. PGP4008 significantly inhibited the function of CX3CR1+ CD8+ T cells in the presence of CP (
To examine whether the frequency of CX3CR1+ Granzyme B+ CD8+ T cells would reflect the therapeutic effects of the CIT, two schedules of CIT were designed, according to the two phases of T cell responses to tumors in an animal model (Liu et al., supra; and Pulko et al. J Immunol, 2011, 187(11):5606-5614). In this model, the frequency of tumor antigen specific effector CD8+ T cells peaked at day 10-14 post tumor inoculation within tumor tissues. According to the kinetics of T cell responses within tumors, the expansion phase was defined as days 7-9 and the effector phase was defined as days 10-14 of the antitumor responses. Anti-PD-1/L1 therapy was given to cover the expansion and effector phases according to the dynamic expression of PD-1 (Pulko et al., supra). Chemotherapy (CP) was given at either phase in order to evaluate its impact on T cell responses (
Since CX3CR1 is a chemokine receptor that is critical for accumulation of T cells at tumor sites (Kee et al., Mol Cin Oncol, 2013, 1(1):35-40), studies were conducted to examine whether the expression of CX3CR1 is required to mediate antitumor activity. Tumor cells were grown in CX3CR1 KO mice, followed by treatment with CIT (Day 10 CP plus anti-PD-1/L1). In contrast to wild type mice, the CIT did not suppress tumor growth in CX3CR1 KO mice (
To address whether the CD8+ T cells specifically require CX3CR1 to mediate antitumor function, adoptive transfer of activated OT-1 CD8+ T cells was performed for treatment of a B16-OVA tumor model. The transfer of CX3CR1+ (but not CX3CR1−) CD8+ T cells significantly suppressed tumor growth (
To determine the effect of chemotherapeutic treatment on survival of CX3CR1+ and CX3CR1− CD8+ T cells, subsets of these cells were isolated, placed in 96 well plates at 2×105 cells/well, and incubated with doxorubicin (Dox) at 0.5 μg/ml for 40 hours. After incubation, T cells were stained with annexin V. T cells affected by Dox were identified as Dox positive cells, and their survival was defined by low binding of annexin V. As shown in
Taken together, the studies described above indicate that CX3CR1 identifies a subset of tumor-reactive CD8+ T cells that can endure chemotherapy and are responsive to PD-1 blockade immunotherapy. The results also indicate that CX3CR1+ CD8+ T cells have at least two advantages allowing them to withstand the toxicity of 15 chemotherapy—drug efflux and downregulation of bmf and ccr5, and may play a key role in clinical responses to combined CIT.
IL-15 has demonstrated antitumor function in preclinical models, especially as a IL-15/IL-15Ra complex that has increased accessibility to T cells in vivo (Stoklasek et al., J Immunol, 2006, 177:6072-6080). For at least a couple of reasons, IL-15 may improve anti-PD-1 therapy for non-responsive tumors. First, the transcription of CD122 (IL-2 receptor beta) was increased in CD11ahigh CD8+ T cells in responders 25 compared to non-responders (
Because CD122 is a component of the IL-15 receptor, it is possible that increased sensitivity to IL-15 causes tumor-reactive CX3CR1+ Granzyme B+ CD8+ T cells to expand beyond the threshold and contribute to tumor rejection in responders, while in the non-responders the CX3CR1+ Granzyme B+ CD8+ T cells might have either lower CD122 expression or lower IL-15 production.
To test whether IL-15 directly contributes to the expansion of CX3CR1+ Granzyme B+ CD8+ T cells, human recombinant IL-15 was incubated for 24 hours with PBMC isolated from healthy human donors, followed by flow cytometry analysis of CX3CR1+ Granzyme B+ CD8+ T cells. IL-15 significantly increased the expansion of CX3CR1+ Granzyme B+ CD8+ T cells among other cells in the PBMC (
To address the mediators of IL-15 in context of its antitumor function, i.t. injection of IL-15/IL-15Ra complex in combination with i.p. injection of PD-1 antibody was evaluated for treatment of B16-OVA melanomas. Although IL-15 (at the experimental dose) alone did not suppress the growth of B16-OVA tumors, and anti-PD-1 alone only partially delayed the tumor growth, the combination of IL-15 and PD-1 antibody significantly suppressed tumor growth (
In additional studies, the effect of IL-15 and anti-PD-1 on CX3CR1+ effector T cells in tumor tissue was examined. B16-OVA melanomas were treated by i.t. injection of anti-PD-1 antibody (G4, 20 μg), soluble IL-15 (sIL-15) complex (mIL-15: 0.1 mg plus IL-15Ra chain: 0.6 mg), or both, for 3 doses on days 7, 10, and 13. The percentage of CX3CR1+ Granzyme B+ cells among CD11a+CD8+ TILs was determined on day 10 after tumor injection, which was 3 days after one dose of the various reagents. As shown in
IL-15 blockade decreased CX3CR1+ effector cells in tumor tissues. B16-OVA tumors were treated with poly IC (PIC) and/or anti-CD40, which demonstrated antitumor activity (
IL-15 also promoted the efficacy of chemotherapy. B16F10 mouse melanoma tumors were treated with carboplatin (40 μg/g) and paclitaxel (10 μg/g) by i.p. injection on day 10 after tumor injection (s.c. 5×105 cells/mouse). Soluble IL-15 (sIL-15) complex (mIL-15: 0.1 mg plus IL-15Ra chain: 0.6 mg) was administered on days 7, 10, and 13 after tumor injection. As shown in
Collectively, these data suggested that PD-1 ICI can restore the antitumor function of pre-existing T cells, if the numbers of pre-existing T cells are not enough to compete rapid growing tumors, IL-15 is needed to expand additional antitumor effector T cells that are expressing CX3CR1 and have the ability move back to tumor site. Thus, in treatment of tumors that are non-responsive to PD-1 ICI therapy, IL-15 is a strong candidate for combination therapy.
Using this model, studies are conducted to determine whether the therapeutic effects of IL-15/PD-1 blockade are attributed to the increase in CX3CR1+ Granzyme B+ CD8+ T cells within tumors or in secondary lymph nodes, and whether the presence of CX3CR1+ Granzyme B+ CD8+ T cells would prevent treated mice from second challenges of same tumors. According to the treatment timing of
To test whether the synergy of IL-15 and PD-1 blockade in treatment of non-responsive tumors also is dependent on the presence of CX3CR1+ CD8+ T cells, tumor models are used (B16F10, LLC) in WT and CX3CR1 KO mice following the same treatment schedule as in
To more specifically address the role of CX3CR1+ CD8+ T cells in mediating the antitumor function of IL-15, studies are conducted to test whether transfer of IL-15 expanded CX3CR1+ Granzyme B+ CD8+ T cells can be used with PD-1 ICI to treat non-responsive tumors. Since IL-15 can selectively expand human CX3CR1+ Granzyme B+ CD8+ T cells in vitro (
PD-1 blockade aims to block the engagement of PD-1 with its ligand PD-L1 in order to restore or enhance T cell function and survival (Dong et al., Nat Med, 2002, 8:793-800; and Iwai et al., Proc Natl Acad Sci USA, 2002, 99:12293-12297). Since none of the molecules in the PD-1 signaling pathway had previously been used to monitor the effects of PD-1 blockade in T cells, signaling molecules in the PD-1/PD-L1 pathway were investigated. These studies revealed that PD-L1 stimulates Bim up-regulation in activated CD8+ T cells as a mechanism for T cell apoptosis (Gibbons et al., Oncoimmunology, 2012, 1:1061-1073), and anti-PD-1 antibodies blocked the Bim up-regulation induced by PD-L1 protein or PD-L1 positive tumors in vitro and in vivo.
Further studies were conducted to examine and compare the frequency of Bim+ cells among circulating CD11ahigh CD8+ T cells, since this population of T cells is enriched with tumor-reactive T cells (Liu et al., Oncoimmunology, 2013, 2:e23972). As shown in
To determine whether the frequency of Bim+ CD8+ T cells would decrease in responders after PD-1 ICI therapy, the % Bim+ CD8+ T cells was examined and compared between responders and non-responders in a small cohort of patients with metastatic melanoma, 12 weeks after anti-PD-1 (pembrolizumab) therapy. Interestingly, it was observed that the % Bim+ CD8+ T cells significantly decreased in responders compared to non-responders at 12 weeks (
Additional studies were carried out to validate this observation in another cohort of patients with metastatic melanoma. Most of the second cohort received PD-1 ICI therapy as first line therapy. Based on patents with clear clinical outcomes, the changes in % Bim+ CD8+ T cells at 12 weeks after PD-1 therapy were examined and compared in complete responders and in non-responders (with disease progression). Interestingly, although most of responders demonstrated a decrease in % Bim+ CD8+ T cells after PD-1 ICI therapy, some non-responders (about 40%) also had a decrease in % Bim+ CD8+ T cells after PD-1 ICI therapy (
The findings discussed in the Examples above indicate a negative correlation between decreased % Bim+ CD8+ T cells and increased CX3CR1+ Granzyme B+ CD8+ T cells in melanoma patients after PD-1 ICI therapy, which would either follow a liner relationship or a curvilinear relationship (
In particular, a prospective bio-specimen collection study is performed in a larger group of male and female patients with metastatic melanoma (about 100 people). This expansion allows a correlation of changes in Bim+ and CX3CR1+ Granzyme B+ CD8+ T cells after PD-1 therapy to be established. Fresh peripheral blood samples (e.g., 60 ml) are collected at baseline (prior to initiation of immunotherapy) and at 12 weeks after PD-1 ICI therapy. The tumor evaluation schedule is done per clinical practice every 6-12 weeks using both RECIST and irRC (Immune Related Response Criteria). Fresh PBMC are stained with antibodies to Bim, CX3CR1, Granzyme B, CD11a, CD8, CD3, and CD45 in the same tube to avoid variables in inter-tube staining of cell surface and intracellular molecules. Live CD45+CD3+ cells are gated followed by sub-gating of CD11ahigh CD8+ T cells, as illustrated in
According to the studies on two cohorts of metastatic melanoma patients treated with anti-PD-1 antibody (
Studies are conducted to show the tumor-reactivity of circulating CX3CR1+ Granzyme B+ CD8+ T cells, establishing these cells as a reliable cellular marker for PD-1 therapy responsiveness. To determine tumor antigen specificity, gp100, tyrosinase, and MART-1 pentamer (ProImmune, Pro5 MHC Class I Pentamers) staining is performed using CX3CR1+ Granzyme B+ CD8+ T cells isolated from HLA-A0201+ patients. Functionally, HLA-A0201+ patient PBMCs are stimulated with pooled melanoma antigen peptides, and IFN-γ production is measured in CX3CR1+ Granzyme B+ CD8+ T cells as described elsewhere (Dronca et al., 2016, JCI Insight 1:e86014; and Romero et al., J Immunol, 2007, 178:4112-4119). To determine disease-specific T cell responses for patients who are not HLA-A0201+, DNA is extracted from CX3CR1+ CD8+ T cells isolated from peripheral blood (using age and gender-matched healthy donors as controls), and analyzed using an ImmunoSeq multiplex PCR assay (Adaptive Biotechnologies), followed by sequencing TCR beta CDR3 to identify and quantify clones of the CX3CR1+ CD8+ T cell subset. Clonal frequency is calculated as the ratio of clonal abundance of all the productive TCR sequences normalized to the number of unique TCR sequences. Since the RNA-seq data showed an increase in TCRVα5 and TCRVβ4-2 among CD11ahigh CD8+ T cells in responders after PD-1 therapy (
Peripheral blood provides a less invasive way to directly assess T cell phenotypes in cancer patients, but there are functional and phenotypic differences between T cells present at the tumor sites and in circulation. To show whether circulating Bim+ or CX3CR1+ CD8+ T cells share similar T cell clones with their counterparts in tumor tissues, tumor biopsies are obtained and analyzed. DNA is extracted from CX3CR1+ Granzyme B+ T cells (sorted by flow cytometry) from peripheral blood and tumor tissues (laser capture for CX3CR1+ Granzyme B+ as shown in
To assess T cell differentiation, proliferation, and function, CX3CR1+ and CX3CR1− CD8+ T cells isolated from melanoma patients are examined and compared before and after PD-1 ICI therapy. The endogenous proliferation of CX3CR1+/− CD8+ T cells is examined by intracellular staining for Ki67, since Ki67+ cells have been identified in tumor-reactive CD8+ T cells in responders to PD-1 IC therapy (Huang et al., Nature, 2017, 545:60-65; and Kamphorst et al., Proc Natl Acad Sci USA, 2017, 114:4993-4998). If CX3CR1+ CD8+ T cells have increased proliferation after PD-1 ICI therapy in responders, further studies are conducted to determine the cytokine that contributes to their proliferation. To that end, CX3CR1+/− CD8+ T cells are labeled with CFSE (an intracellular dye for cell division), and cultured with graded concentration of IL-2, IL-7, or IL-15 for 11 days. If spontaneous proliferation is not observed by day 5, the cells are removed to new culture wells containing anti-CD3/CD28 beads to initiate T cell proliferation with fresh cytokines. After incubation, the proportion of proliferative cells (CFSE dilution) between these two subsets is measured. In addition, studies are conduced to confirm whether cytokine receptor expression is different between CX3CR1+ CD8+ T cells and CX3CR1− CD8+ T cells, or between responders ad non-responders, since transcription of CD122 (IL-2/IL-15Rβ) was increased in responders as compared to non-responders after PD-1 ICI (
CTL function (CD107a, Granzyme B, and perforin) and intracellular production of IFN-γ, TNF-α and IL-2 are examined ex vivo. To determine tumor antigen-induced function, PBMC are stimulated with pooled melanoma antigen peptides, and IFN-γ production is measured in CX3CR1+/− CD8+ T cells as described elsewhere (Dronca et al., JCI Insight, 2016, 1: e86014; and Romero et al., J Immunol, 2007, 178:4112-4119). PBMC from patients who are not HLA-A0201+ are stimulated with anti-CD3/CD28 beads to trigger their CTL function.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims benefit of priority from U.S. Provisional Application Ser. No. 62/641,672, filed on Mar. 12, 2018. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/21802 | 3/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62641672 | Mar 2018 | US |
