Soluble CD28 levels during immunotherapy

Abstract

To provide methods of diagnosing cancer relapse in a subject and of predicting imminent cancer relapse in the subject.SOLUTION: There are provided methods of diagnosing and predicting cancer relapse comprising measuring soluble CD28 levels in a subject, where an increase in soluble CD28 is indicative of cancer relapse or imminent cancer relapse. There are also provided methods of determining response to PD-1 / PD-L1 based immunotherapy in a subject in need thereof comprising measuring sCD28 levels in a subject undergoing PD-1 / PD-L1 based immunotherapy at least at two points in time, where a decrease indicates response and an increase indicates lack of response.SELECTED DRAWING: Figure 2D

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 942,276, filed December 2, 2019, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present invention is in the field of immunomodulation and immunotherapy. [Background technology]

[0003] Some costimulatory molecules are known to have several physiological forms. In addition to membrane-bound forms, soluble forms expressed in naive immune cells have also been reported, adding to the complexity of T cell biology. The soluble form of CD28 (sCD28) has been implicated in the inhibition of immune responses to cancer. In addition, sCD28 has been demonstrated to suppress the efficacy of PD-1 and PD-L1-based immunotherapy. It has been hypothesized that the presence of sCD28 may be a factor contributing to the heterogeneity of responses to immunotherapy.

[0004] However, the status of sCD28 during the course of immunotherapy and its possible role in relapse have not been investigated. Furthermore, the role of sCD28 levels in patients who become resistant to immunotherapy treatment is unclear. Summary of the Invention

[0005] The present invention provides a method for diagnosing cancer recurrence in a subject and predicting impending cancer recurrence in a subject, the method comprising measuring soluble CD28 (sCD28) levels in the subject, where an increase in sCD28 levels indicates that the subject is recurring or that recurrence is imminent. Also provided is a method for determining response to PD-1 / PD-L1-based immunotherapy in a subject in need thereof, the method comprising measuring sCD28 levels in a subject receiving PD-1 / PD-L1-based immunotherapy at least two time points, where a decrease indicates a response and an increase indicates a lack of response.

[0006] According to a first aspect, there is provided a method of determining response to PD-1 / PD-L1 based immunotherapy in a subject suffering from cancer, the method comprising measuring sCD28 levels in the subject at least two time points, at least one of which is following initiation of PD-1 / PD-L1 based immunotherapy, wherein an increase in sCD28 levels from the first time point to the second time point following initiation of PD-1 / PD-L1 based immunotherapy indicates that the subject is not a responder to immunotherapy, and a decrease or no change in sCD28 levels from the first time point to the second time point following initiation of PD-1 / PD-L1 based immunotherapy indicates that the subject is a responder to immunotherapy, thereby determining the response to PD-1 / PD-L1 based immunotherapy in the subject.

[0007] According to some embodiments, the first time point is prior to or at the start of immunotherapy.

[0008] According to some embodiments, the first time point is after initiation of immunotherapy.

[0009] According to some embodiments, the second time point is at least 6 weeks after the first time point.

[0010] According to some embodiments, the second time point is up to 7 weeks after the first time point.

[0011] According to some embodiments, a. the first time point is at or before the initiation of immunotherapy and the second time point is 7 weeks or 13 weeks after the initiation of immunotherapy; or b. The first time point is 7 weeks after the initiation of immunotherapy and the second time point is 13 weeks after the initiation of immunotherapy.

[0012] According to some embodiments, a decrease in sCD28 levels from the first time point to the second time point after initiation of PD-1 / PD-L1-based immunotherapy indicates that the subject is a responder to the immunotherapy.

[0013] According to some embodiments, the decrease is a decrease in sCD28 of at least 1 ng / ml.

[0014] According to another aspect, there is provided a method of diagnosing cancer recurrence in a subject in need thereof, the method comprising measuring soluble CD28 (sCD28) levels in the subject, where an increase in sCD28 levels in the subject is indicative of recurrence, thereby diagnosing cancer recurrence in the subject.

[0015] According to some embodiments, the method of the present invention comprises: a. Discontinuing immunotherapy in non-responder subjects; and b. administering a different immunotherapy to subjects who are non-responders; and c. continuing to administer the PD-1 / PD-L1 based immunotherapy to subjects who are responders; and d. Increasing the dose of PD-1 / PD-L1 based immunotherapy administered to subjects who are responders.

[0016] According to another aspect, there is provided a method of predicting cancer recurrence in a subject in need thereof, the method comprising measuring soluble CD28 (sCD28) levels in the subject, wherein an increase in sCD28 levels in the subject indicates impending cancer recurrence, thereby predicting cancer recurrence in the subject.

[0017] According to some embodiments, the subject has cancer and the cancer is in remission.

[0018] According to some embodiments, the subject previously had cancer and is currently cancer-free.

[0019] According to some embodiments, the subject has undergone or is undergoing immunotherapy.

[0020] According to some embodiments, the immunotherapy is PD-1, PD-L1 and / or CD80-based immunotherapy.

[0021] According to some embodiments, the increase is compared to sCD28 levels in the subject prior to relapse.

[0022] According to some embodiments, the increase is compared to a pre-defined threshold.

[0023] According to some embodiments, the increase is at least a 50% increase.

[0024] According to some embodiments, the increase is to at least 6 ng / mL of sCD28.

[0025] According to some embodiments, the increase is an increase in sCD28 of at least 1 ng / ml.

[0026] According to some embodiments, the measuring comprises obtaining a sample from the subject and measuring the level of sCD28 in the sample.

[0027] In some embodiments, the sample is a blood sample.

[0028] According to some embodiments, measuring includes measuring at multiple time points, at least one time point at which the subject is known to be in remission or cancer-free, and at least one time point at which the subject is at risk of recurrence.

[0029] According to some embodiments, remission includes partial and complete responses to the therapy.

[0030] According to some embodiments, at the time the subject is known to be in remission or cancer-free, sCD28 levels are below 5 ng / mL.

[0031] According to some embodiments, the subject has a blood sCD28 level of less than 6 ng / mL prior to carrying out the method.

[0032] According to some embodiments, the cancer is selected from skin cancer, urothelial cancer, lung cancer, and renal cancer.

[0033] According to some embodiments, the cancer is selected from melanoma and urothelial cancer.

[0034] According to some embodiments, the urgency is within the next 20 weeks.

[0035] According to some embodiments, the methods of the invention further comprise administering another immunotherapy to the subject diagnosed with or predicted to have a cancer recurrence.

[0036] According to some embodiments, the methods of the invention further comprise administering to the subject determined to be a non-responder to the immunotherapy another immunotherapy.

[0037] According to some embodiments, the immunotherapy comprises: a. checkpoint inhibitors, b. Chimeric antigen receptor (CAR)-based therapies, and c. Selected from cancer vaccines.

[0038] According to some embodiments, the checkpoint inhibitor is a PD-1 and / or PD-L1 based immunotherapy.

[0039] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0040] [Figure 1] Survival chart for melanoma patients receiving anti-PD1 therapy. Patients with low levels of sCD28 before starting therapy had a longer median survival time than patients with high levels of sCD28. [Figure 2A-D] (2A) Line graph of sCD28 levels in 17 patients (9 responders and 8 non-responders) over the course of several weeks of immunotherapy. (2B and 2C) Bar graphs of mean sCD28 levels in (2B) non-responders and (2C) responders to immunotherapy at three time points. (2D) Bar graph of absolute change in sCD28 expression from the first reading before initiation of immunotherapy to two time points after initiation. *, Pval=0.06, **, Pval<0.05 by signed rank test. [Diagram 3] Line graph of sCD28 levels in two patients who responded to immunotherapy and then experienced a recurrence of cancer. CR is complete response, PR is partial response, PD is progressive disease, and SD is stable disease. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention provides, in some embodiments, methods for diagnosing and predicting cancer recurrence in a subject who has or still has cancer, comprising measuring soluble CD28 (sCD28 levels) in the subject, where an increase in sCD28 indicates cancer recurrence or impending cancer recurrence. The present invention further provides a method for determining response to PD-1 / PD-L1-based immunotherapy by measuring changes in sCD28 levels during the course of immunotherapy treatment. The present invention is based on the surprising discovery that in subjects who have responded to immunotherapy, spikes in sCD28 levels act as a marker of recurrence that has begun or is about to begin. Furthermore, the present invention is based on the surprising discovery that sCD28 levels increase in subjects not responding to immunotherapy, and do not change or decrease in subjects who are responding.

[0042] According to a first aspect, there is provided a method of diagnosing cancer recurrence in a subject in need thereof, the method comprising measuring soluble CD28 (sCD28) levels in the subject, wherein an increase in sCD28 levels in the subject is indicative of cancer recurrence, thereby diagnosing cancer recurrence.

[0043] According to another aspect, there is provided a method of predicting cancer recurrence in a subject in need thereof, the method comprising measuring sCD28 levels in the subject, wherein an increase in sCD28 levels in the subject is indicative of upcoming cancer recurrence.

[0044] According to another aspect, there is provided a method of determining response to PD-1 / PD-L1 based immunotherapy in a subject in need thereof, the method comprising measuring sCD28 levels in the subject at least two time points, where an increase in sCD28 levels indicates that the subject is not responding to or is a responder to the immunotherapy and a decrease or no change in sCD28 levels indicates that the subject is responding to or is a responder to the immunotherapy, and determining the response to PD-1 / PD-L1 based immunotherapy.

[0045] In some embodiments, the CD28 is a mammalian CD28. In some embodiments, the CD28 is human CD28. In some embodiments, the human CD28 comprises or consists of the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 1). In some embodiments, the mature CD28 lacks the signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 2).

[0046] In some embodiments, the DNA coding sequence encoding full length human CD28 comprises the sequence: (SEQ ID NO:3).

[0047] As used herein, sCD28 refers to any CD28 fragment or variant that does not contain a transmembrane domain and therefore cannot be integrated into a membrane. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 4). In some embodiments, sCD28 is not membrane bound. In some embodiments, sCD28 is in solution. In some embodiments, sCD28 is CD28 in blood. In some embodiments, sCD28 is CD28 in TME. In some embodiments, sCD28 is CD28 in body fluids. In some embodiments, sCD28 is a cleavage product from membrane CD28 (mCD28). In some embodiments, sCD28 is a truncated CD28. In some embodiments, sCD28 lacks the cytoplasmic domain of full-length CD28. In some embodiments, sCD28 is a dimeric sCD28. In some embodiments, sCD28 is a monomeric sCD28. In some embodiments, sCD28 is not a splice variant resulting from alternative splicing of CD28. In some embodiments, sCD28 comprises the amino acid sequence: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO: 5). In some embodiments, sCD28 lacks the signal peptide and comprises the sequence: NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP (SEQ ID NO: 6).

[0048] In some embodiments, the subject previously had cancer. In some embodiments, the subject still has cancer. In some embodiments, the subject has cancer. In some embodiments, the subject had cancer but is now cancer-free. In some embodiments, the subject has cancer that is in remission. In some embodiments, the subject has cancer that is in partial remission. In some embodiments, remission includes complete remission and partial remission. In some embodiments, remission includes partial response and complete response. In some embodiments, remission includes complete remission. In some embodiments, remission includes partial remission. In some embodiments, the subject has undergone therapy for cancer. In some embodiments, the subject is undergoing therapy for cancer. In some embodiments, the therapy is immunotherapy. In some embodiments, the subject is a responder to the therapy. In some embodiments, the subject is a non-responder to the therapy. In some embodiments, the subject has undergone PD-1 / PD-L1 based immunotherapy. In some embodiments, the subject is undergoing PD-1 / PD-L1 based immunotherapy.

[0049] In some embodiments, a responder is a subject that responds to a therapy. In some embodiments, a responder is a person who is responding. In some embodiments, a person who is responding is a responder. In some embodiments, a responder is a subject that responds favorably to a therapy. A "favorable response" of a cancer patient indicates the "responsiveness" of a cancer patient to treatment with a therapy, i.e., treatment of a responsive cancer patient with immunotherapy results in a desirable clinical outcome, such as tumor regression, tumor shrinkage or tumor necrosis, an anti-tumor response by the immune system, and prevents or delays tumor recurrence, tumor growth, or tumor metastasis. In some embodiments, the response is a complete response (CR). In some embodiments, the response is a partial response (PR). In some embodiments, the response is a regression of the cancer. In some embodiments, the regression is from stable disease. In some embodiments, the regression is from progressive disease. In some embodiments, a non-responder is a subject that does not respond to a therapy. In some embodiments, a non-responder is a person who is not responding. In some embodiments, a person who is not responding is a non-responder. In some embodiments, a non-responder is a subject that responds unfavorably to a therapy. As used herein, an "unfavorable response" of a cancer patient refers to the "non-responsiveness" of a cancer patient to treatment with a therapy, and thus treatment of a non-responsive cancer patient with a therapy does not result in a desired clinical outcome and may result in undesirable outcomes, such as clinical outcomes and tumor expansion, recurrence and metastasis. In some embodiments, the undesirable outcome is cancer recurrence. In some embodiments, the undesirable outcome is the development of stable disease. In some embodiments, the undesirable outcome is the development of progressive disease.

[0050] As used herein, the terms "complete response", "partial response", "stable disease" and "progressive disease" are all criteria for evaluating cancer, particularly solid lesions. During evaluation, lesions are measured to provide a basis for comparison and evaluation during treatment. In some embodiments, a complete response refers to all lesions that have disappeared over the course of treatment. In some embodiments, a complete response refers to all target lesions that have disappeared over the course of treatment. In some embodiments, a partial response refers to a reduction in size of one or more lesions. In some embodiments, the reduction in size is of the largest lesion. In some embodiments, the reduction is in the sum of the sizes of all lesions. In some embodiments, the reduction is in the average size of the lesions. In some embodiments, the reduction in size is at least a 10, 20, 30, 40, or 50% reduction. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction in size is at least a 30% reduction. In some embodiments, the reduction is a reduction in the diameter of the lesion. In some embodiments, stable disease refers to a significant reduction or no increase in the size of one or more lesions. In some embodiments, progressive disease refers to an increase in size of one or more lesions. In some embodiments, the increase is in the largest lesion. In some embodiments, the increase is in the sum of the size of all lesions. In some embodiments, the increase is in the average size of the lesions. In some embodiments, the increase in size is at least a 10, 20, 30, 40, or 50% increase. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase in size is at least a 20% increase.

[0051] In some embodiments, the immunotherapy is PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the immunotherapy is PD-1 based immunotherapy. In some embodiments, the immunotherapy is PD-L1 based immunotherapy. In some embodiments, the PD-1 / PD-L1 based immunotherapy comprises administering an anti-PD1 or anti-PD-L1 antibody. In some embodiments, the therapy comprises blockade of the PD-1 checkpoint. In some embodiments, the immunotherapy comprises administering allogeneic, syngeneic, or autologous immune cells to the subject. In some embodiments, the immune cells are T cells. In some embodiments, the subject in need of immunotherapy is afflicted with cancer. In some embodiments, the PD-1 based immunotherapy comprises PD-L2 based immunotherapy. In some embodiments, the PD-L1 based immunotherapy comprises PD-L2 based immunotherapy.

[0052] In some embodiments, the immunotherapy is CD80-based immunotherapy. In some embodiments, the immunotherapy is CD86-based immunotherapy. CD80 and CD86 immunotherapy are well known in the art and include administering CD80 / CD86 and / or mimetics, derivatives or mimetics thereof to stimulate an immune response. CD80-Fc, as a non-limiting example, is currently in clinical trials as an anti-cancer immunotherapy.

[0053] In some embodiments, the immunotherapy is a CTLA-4 based immunotherapy. In some embodiments, the CTLA-4 immunotherapy comprises CTLA-4 blockade. In some embodiments, the CTLA-4 immunotherapy comprises administering a CTLA-4 inhibitor. In some embodiments, the CTLA-4 immunotherapy comprises administering an anti-CTLA-4 antibody. In some embodiments, the therapy comprises blockade of the CTLA-4 checkpoint. In some embodiments, the immunotherapy is a combination therapy of any of the immunotherapies described herein.

[0054] In some embodiments, the subject is afflicted with cancer. In some embodiments, the cancer is a cancer that can be treated with immunotherapy. In some embodiments, the cancer is a cancer that can be treated with PD-1 / PD-L1 therapy. In some embodiments, the subject has received PD-1 / PD-L1 therapy. In some embodiments, the subject is a non-responder to PD-1 / PD-L1 therapy. In some embodiments, the subject is naive to PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are performed in conjunction with PD-1 / PD-L1 therapy. In some embodiments, the methods of the invention are performed prior to PD-1 / PD-L1 therapy.

[0055] In some embodiments, the cancer is a cancer with elevated sCD28 levels. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are at and / or above 5, 6, 7, 8, 9, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 ng / mL. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer comprises high sCD28 levels. In some embodiments, the elevated and / or high sCD28 levels are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% and / or more than the levels in healthy subjects. Each possibility represents a separate embodiment of the invention. In some embodiments, the cancer is not breast cancer. In some embodiments, the cancer is selected from melanoma, urothelial cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from melanoma, urothelial cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, and colorectal cancer. In some embodiments, the cancer is melanoma, urothelial cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, renal cancer, gastric cancer, or colorectal cancer. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer is selected from melanoma and urothelial cancer. In some embodiments, the cancer is selected from skin cancer, urothelial cancer, lung cancer, and renal cancer. In some embodiments, the cancer is selected from skin cancer and urothelial cancer.

[0056] In some embodiments, sCD28 is measured at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 time points. Each possibility represents a separate embodiment of the present invention. In some embodiments, sCD28 is measured at least two time points. In some embodiments, the at least two time points include a first time point and a second time point. In some embodiments, there is an increase from one time point to the second time point. In some embodiments, there is a decrease from one time point to the second time point. In some embodiments, there is no change from one time point to another time point. In some embodiments, there is a trend in sCD28 kinetics. In some embodiments, the trend is an increase or a decrease. In some embodiments, an increasing trend indicates that the subject is not responding to the therapy. In some embodiments, an increasing trend indicates cancer recurrence. In some embodiments, an increasing trend indicates impending cancer recurrence.

[0057] In some embodiments, the subject that responds to therapy is a responder. In some embodiments, the subject that does not respond to therapy is a non-responder. The response to therapy does not have to be a complete response, but can be a partial response, or a response of improvement of at least one symptom. In some embodiments, a non-responder does not have a positive response to therapy.

[0058] In some embodiments, the increase is compared to sCD28 levels in a healthy subject. In some embodiments, the increase is compared to sCD28 levels in a subject prior to relapse. In some embodiments, the increase is compared to sCD28 levels in a subject after initiation of immunotherapy. In some embodiments, the increase is compared to sCD28 levels after disease remission. In some embodiments, the increase is compared to a pre-defined threshold. In some embodiments, the increase is compared to levels prior to initiation of immunotherapy.

[0059] In some embodiments, the increase is from one time point to another. In some embodiments, the increase is from a first time point to a second time point. In some embodiments, the increase indicates that the subject is a non-responder to the therapy. In some embodiments, the increase indicates that the subject is not responding to the therapy. In some embodiments, the method further comprises discontinuing therapy for non-responding patients. In some embodiments, the method further comprises increasing the dose for non-responding patients. In some embodiments, the increase is a statistically significant increase.

[0060] In some embodiments, at least one time point is at or before the initiation of treatment. In some embodiments, at least one time point is after the initiation of treatment. In some embodiments, the first time point is before therapy. In some embodiments, the first time point is at the initiation of therapy. In some embodiments, the first time point is in remission. In some embodiments, the first time point is when the subject is cancer-free. In some embodiments, the first time point is before the initiation of immunotherapy. In some embodiments, the first time point is at the initiation of immunotherapy. In some embodiments, the first time point is during immunotherapy. In some embodiments, the second time point is a current measurement. In some embodiments, the second time point is a testing time point. In some embodiments, the second time point is a time point when the subject is at risk of recurrence. In some embodiments, the second time point is after the initiation of immunotherapy. In some embodiments, the second time point is during immunotherapy. In some embodiments, the first time point is before the initiation of immunotherapy and the second time point is after the initiation of immunotherapy. In some embodiments, the first and second time points are after initiation of immunotherapy.

[0061] In some embodiments, the first and second time points are at least 1, 2, 3, 4, 5, 6, or 7 weeks apart. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first and second time points are at least 6 weeks apart. In some embodiments, the first and second time points are at least 7 weeks apart. In some embodiments, the first and second time points are at most 1, 2, 3, 4, 5, 6, or 7 weeks apart. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first and second time points are at most 6 weeks apart. In some embodiments, the first and second time points are at most 7 weeks apart. In some embodiments, the first and second time points are about 6 weeks apart. In some embodiments, the first and second time points are about 7 weeks apart. In some embodiments, the first and second time points are about 6-7 weeks apart. In some embodiments, the first and second time points are 6-7 weeks apart. In some embodiments, the second time point is 7 weeks after the start of immunotherapy. In some embodiments, the first time point is at the start of immunotherapy. In some embodiments, the first time point is 7 weeks after the start of immunotherapy. In some embodiments, the second time point is 13 weeks after the start of immunotherapy. In some embodiments, the second time point is 7 weeks or 13 weeks after the start of immunotherapy.

[0062] In some embodiments, the increase is at least a 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000% increase. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is from no expression to expression of sCD28. In some embodiments, the increase is from the absence of sCD28 to the presence of sCD28. In some embodiments, the increase is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL of sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is at least 1 ng / mL of sCD28. In some embodiments, the increase is at least 2 ng / mL of sCD28. In some embodiments, the increase is at least 5 ng / mL of sCD28. In some embodiments, the increase is at least 6 ng / mL of sCD28. In some embodiments, the increase is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL of sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is at least 1 ng / mL of sCD28. In some embodiments, the increase is at least 2 ng / mL of sCD28.

[0063] In some embodiments, a decrease in sCD28 levels from a first time point to a second time point indicates that the subject is a responder to immunotherapy. In some embodiments, a change in sCD28 levels from a first time point to a second time point indicates that the subject is a responder to immunotherapy. In some embodiments, an increase in sCD28 levels from a first time point to a second time point indicates that the subject is a non-responder to immunotherapy. In some embodiments, the decrease is from one time point to another. In some embodiments, the decrease is from a first time point to a second time point. In some embodiments, the decrease indicates that the subject is a responder to the therapy. In some embodiments, the decrease indicates that the subject is responding to the therapy. In some embodiments, the method further comprises continuing therapy to the responding patient. In some embodiments, the method further comprises increasing therapy to the responding patient. In some embodiments, the decrease is a statistically significant decrease. In some embodiments, the method comprises discontinuing therapy to the non-responding patient. In some embodiments, the method comprises administering a different therapy to the non-responding patient. In some embodiments, the different therapy is another therapy.

[0064] In some embodiments, there is no change from one time point to another. In some embodiments, the no change is from a first time point to a second time point. In some embodiments, the no change indicates that the subject is a responder to the therapy. In some embodiments, the no change indicates that the subject is responding to the therapy. In some embodiments, the change comprises a change that is not statistically significant. In some embodiments, the decrease is at least a 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99, or 100% decrease. Each possibility represents a separate embodiment of the invention. In some embodiments, the decrease is to no expression of sCD28. In some embodiments, the decrease is to substantially no expression of sCD28. In some embodiments, the decrease is to less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL of sCD28. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction is less than 2 ng / mL of sCD28. In some embodiments, the reduction is less than 5 ng / mL of sCD28. In some embodiments, the reduction is less than 6 ng / mL of sCD28. In some embodiments, the reduction is at least 1, 2, 3, 4, or 5 ng / mL of sCD28. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is at least 1 ng / mL of sCD28. In some embodiments, the reduction is at least 2 ng / mL of sCD28.

[0065] In some embodiments, the subject's blood contains high levels of sCD28. In some embodiments, the increase is to elevated levels. In some embodiments, the levels are elevated above levels in healthy subjects. In some embodiments, the subject's sCD28 levels are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% elevated above levels in healthy subjects. Each possibility represents a separate embodiment of the present invention. In some embodiments, the levels are elevated to greater than 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng / mL of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the levels are elevated to greater than 5 ng / mL. In some embodiments, the levels are elevated to greater than 10 ng / mL. In some embodiments, the levels are elevated to greater than 20 ng / mL. In some embodiments, the subject's blood comprises at least 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng of sCD28 per mL of blood. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood comprises at least 5 ng / mL of sCD28. In some embodiments, the subject's blood comprises at least 10 ng / mL of sCD28. In some embodiments, the subject's blood contains at least 20 ng / mL of / sCD28.

[0066] In some embodiments, the subject's blood comprises a healthy level of sCD28 prior to recurrence. In some embodiments prior to recurrence, the subject's blood comprises a level of sCD28 that is not elevated prior to recurrence. In some embodiments, the subject's blood comprises less than at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 ng / mL of sCD28 prior to recurrence. In some embodiments, the subject's blood comprises less than 5 ng / mL of sCD28 prior to recurrence. In some embodiments, the subject's blood comprises less than 6 ng / mL of sCD28 prior to recurrence. Each possibility represents a separate embodiment of the present invention. In some embodiments, the subject's blood is the subject's blood prior to performing the methods of the present invention.

[0067] In some embodiments, measuring comprises obtaining a sample from the subject. In some embodiments, measuring comprises measuring sCD28 levels in the sample. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a bodily fluid. In some embodiments, the sample is blood. In some embodiments, the sample is from a subject. In some embodiments, the sample comprises a bodily fluid. In some embodiments, the sample comprises a tissue. In some embodiments, the sample comprises a cell. In some embodiments, the detection is by a secondary antibody. In some embodiments, the detection is a labeled molecule that binds to sCD28. In some embodiments, the detection is by ELISA. In some embodiments, the detection is by immunohistochemistry. In some embodiments, the detection is by immunoblot. Agents for detecting sCD28 are described in International Patent Application WO2019 / 175885, which is incorporated herein by reference in its entirety.

[0068] In some embodiments, measuring comprises measuring at multiple time points. In some embodiments, the multiple time points are at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 time points. Each possibility represents a separate embodiment of the invention. In some embodiments, the multiple time points are two time points. In some embodiments, the multiple time points are at least two time points. In some embodiments, the multiple time points include a first and a second time point. In some embodiments, at least one time point is a time point at which the subject is in remission or cancer-free. In some embodiments, at least one time point is a time point at which the subject is known to be in remission or cancer-free. In some embodiments, the time point at which the subject is in remission or cancer-free or known to be in remission or cancer-free is a time point at which the subject has an sCD28 level of or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL of sCD28. Each possibility represents a separate embodiment of the invention. In some embodiments, the time point is when the subject has an sCD28 level of less than 2 ng / mL. In some embodiments, the time point is when the subject has an sCD28 level of 5 ng / mL or less. In some embodiments, the time point is when the subject has an sCD28 level of 6 ng / mL or less. In some embodiments, at least one time point is after initiation of therapy.

[0069] In some embodiments, at least one time point is a time point when the subject is at risk of recurrence. In some embodiments, at least one time point is a time point when the subject is suspected of recurrence. In some embodiments, at least one time point is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 weeks after the onset of recurrence. Each possibility represents a separate embodiment of the invention. In some embodiments, at least one time point is at least 35 weeks after the onset of therapy. In some embodiments, at least one time point is at least 40 weeks after the onset of therapy. In some embodiments, at least one time point is at least 45 weeks after the onset of therapy. In some embodiments, at least one time point is at least 46 weeks after the onset of therapy.

[0070] In some embodiments, the upcoming recurrence is an imminent recurrence. In some embodiments, the imminent recurrence is a recurrence. In some embodiments, the imminent is within the next 10, 15, 20, 25, 30, 35, or 40 weeks. Each possibility represents a separate embodiment of the present invention. In some embodiments, the upcoming recurrence is a recurrence at any time in the future. In some embodiments, the imminent recurrence is within the next 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 weeks. Each possibility represents a separate embodiment of the present invention.

[0071] In some embodiments, the method further comprises administering another therapy to the subject. In some embodiments, the another therapy is a different therapy. In some embodiments, the another therapy is a therapy that treats cancer. In some embodiments, the method further comprises administering another therapy to a subject diagnosed with or predicted to have cancer recurrence. In some embodiments, the method further comprises administering another therapy to a subject diagnosed with cancer recurrence. In some embodiments, the method further comprises administering another therapy to a subject predicted to have cancer recurrence. In some embodiments, the method further comprises administering another therapy to a subject determined to be a non-responder. In some embodiments, the method further comprises administering another therapy to a subject determined to be a non-responder to immunotherapy. In some embodiments, the other therapy is not the therapy that caused the remission. In some embodiments, the other therapy is a different therapy than the therapy originally used to treat the cancer. In some embodiments, the other therapy is not a PD-1 / PD-L1 based immunotherapy. In some embodiments, the other therapy is not an immunotherapy. In some embodiments, the other therapy is not a therapy to which the subject is a non-responder.

[0072] In some embodiments, the other therapy is an immunotherapy. In some embodiments, the other therapy is a therapy to treat cancer. In some embodiments, the other therapy is a PD-1 and / or PD-L1 based immunotherapy. In some embodiments, the other immunotherapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1 and / or PD-L1 inhibitor. In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the other immunotherapy is a chimeric antigen receptor (CAR) based immunotherapy. In some embodiments, the CAR is a CAR-T. In some embodiments, the CAR is a CAR-NK. In some embodiments, the other immunotherapy is a cancer vaccine.

[0073] As used herein, the terms "administer", "administration" and the like refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of an agent of the present invention to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal.

[0074] As used herein, the terms "CAR-T cells" and "CAR-NK cells" refer to engineered receptors that have specificity for at least one protein of interest (e.g., an immunogenic protein whose expression is increased following treatment with an epigenetic modifier) ​​and are grafted onto immune effector cells (T cells or NK cells). In some embodiments, the CAR-T cells have the specificity of a monoclonal antibody grafted onto a T cell. In some embodiments, the CAR-NK cells have the specificity of a monoclonal antibody grafted onto a NK cell. In some embodiments, the T cells are selected from cytotoxic T lymphocytes and regulatory T cells.

[0075] CAR-T and CAR-NK cells and their vectors are well known in the art. Such cells target proteins that are bound by receptors and exhibit cytotoxicity. In some embodiments, CAR-T or CAR-NK cells target at least one viral protein. In some embodiments, CAR-T or CAR-NK cells target multiple viral proteins. In some embodiments, CAR-T or CAR-NK cells target viral proteins that are increased in expression by contact with epigenetic modifiers.

[0076] The construction of CAR-T cells is well known in the art. In one non-limiting example, monoclonal antibodies against viral proteins can be made, and then vectors encoding the antibodies are constructed. The vectors also include costimulatory signal regions. In some embodiments, the costimulatory signal regions include intracellular domains of known T cell or NK cell stimulatory molecules. In some embodiments, the intracellular domains are selected from at least one of CD3Z, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. In some embodiments, the vectors also include CD3Z signaling domains. This vector is then transfected into T cells, for example, by lentiviral infection.

[0077] As used herein, the term "treatment" or "treating" of a disease, disorder, or condition includes alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, the compositions useful herein only need to reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0078] As used herein, the term "recurrence" refers to a worsening of cancer after a period of improvement. Recurrence does not necessarily mean that the cancer has returned to pre-treatment levels, but only that the cancer has worsened in some way since it was in remission. In some embodiments, recurrence includes the recurrence of cancer after the subject is cancer-free. In some embodiments, recurrence includes continued growth of cancer. In some embodiments, recurrence includes growth of cancer after growth has stopped. In some embodiments, recurrence includes an increase in the rate of cancer growth. In some embodiments, recurrence includes the return of cancer symptoms rather than stopping. In some embodiments, recurrence includes worsening of cancer symptoms rather than improving. Recurrence need only be an increase in the severity of the cancer, or the severity of symptoms associated with the cancer.

[0079] In some embodiments, the recurrence is a recurrent cancer. In some embodiments, the recurrence is refractory to the therapy that caused the remission. In some embodiments, the recurrence is non-responsive to the therapy that caused the remission.

[0080] In some embodiments, the method is performed in vivo. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the measuring is performed ex vivo. In some embodiments, the measuring is performed in vitro. In some embodiments, the sample is an in vitro sample. In some embodiments, the sample is an ex vivo sample.

[0081] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0082] It should be noted that, as used herein and in the appended claims, the singular forms "," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes a plurality of such polynucleotides, a reference to "the polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should further be noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for using exclusive language, such as "sole," "solely," and the like, in connection with reciting claim elements or using "negative" limitations.

[0083] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). It will be further understood by those of ordinary skill in the art that virtually any disjunction and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0084] It is understood that certain features of the invention described in the context of separate embodiments for clarity can also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity can also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and their elements are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination were individually and explicitly disclosed herein.

[0085] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0086] Various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below find experimental evidence in the following examples. EXAMPLES

[0087] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, see, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference. Other general references are provided throughout the book.

[0088] material and method Detection of soluble human CD28 in plasma of cancer patients - Plasma samples from cancer patients with various indications who received anti-PD1 therapy were diluted 1:10 and analyzed for soluble human CD28 by functional ELISA. Samples with high sCD28 were analyzed again at the appropriate dilution. Samples were only examined if clinical observations regarding response to treatment were available. Examples were selected by the criterion of >0.5-fold change in the amount of sCD28 only during treatment.

[0089] Example 1: Pre-immunotherapy sCD28 levels predict response The immunosuppressive effect of sCD28 has already been established (International Patent Application No. 2019 / 175885, incorporated herein by reference in its entirety). Furthermore, sCD28 is a potent inhibitor of immunotherapy, and sCD28 levels have been found to be elevated in many cancers. Anti-PD1 / anti-PD-L1 and CD80-based immunotherapies have all been shown to be adversely affected by sCD28.

[0090] To further understand the impact of sCD28 levels on cancer progression during immunotherapy, over 100 serum samples from melanoma patients prior to initiation of anti-PD1 therapy (Nivolumab, NCT01176461) were tested for sCD28 levels by ELISA. Survival was monitored throughout therapy and survival functions were plotted comparing subjects with high versus low levels of sCD28 prior to therapy (Figure 1). Subjects with low levels of sCD28 (<2ng / mL) were found to have a longer median survival than subjects with high levels (659 days vs. 514 days).

[0091] Example 2: sCD28 levels during immunotherapy Sera from 166 individual patients (melanoma, renal cell carcinoma, lung squamous cell carcinoma and urothelial carcinoma patients) receiving immunotherapy (anti-PD-1, e.g., nivolumab and pembrolizumab, anti-PD-L1, e.g., atezolizumab and anti-CTLA-4, e.g., ipilimumab) were examined for their sCD28 levels. 154 of these 166 provided multiple samples during therapy. Thirty-seven of these subjects were considered positive for sCD28 (>2ng / mL) at at least one time point measured. Nineteen of those 37 showed change kinetics of sCD28 occurrence during therapy (more than 50% change in sCD28 levels). Interestingly, the direction of change (positive of negative kinetics) was always consistent: responders showed a decrease in sCD28 levels and non-responders showed an increase in sCD28 levels (Figure 2A).

[0092] Of these 166 patients, 92 patients had received anti-PD-1 therapy with nivolumab (NCT01176461). All patients provided samples at three time points (baseline, week 7, and week 13) throughout the entire course of immunotherapy. At the end of the study, patients were stratified into responders and non-responders based on cancer remission as measured by tumor size. The impact of sCD28 as a prognostic marker is immediately evident when trends in sCD28 levels are examined across the responder and non-responder populations as a whole. Non-responders showed an increase in sCD28 levels from baseline (pre-therapy) to week 7 and week 13 (Figure 2B). In contrast, responders not only did not show an increase in levels of sCD28, but in fact, the mean sCD28 levels decreased from baseline to week 7 and week 13 (Figure 2C). When the absolute changes in sCD28 expression were quantified at weeks 7 and 13, the increases seen in the non-responder population were statistically significant (Figure 2D).

[0093] Example 3: sCD28 levels during relapse Two of the monitored patients who showed changes in sCD28 dynamics showed unexpected results. Both patients responded to immunotherapy, the first patient (urothelial carcinoma) showed a complete response (CR) and the other patient (melanoma) showed a partial response (PR). Both patients were negative for sCD28 on average during their response to therapy, i.e., during the remission phase (Figure 3). However, both patients showed a significant increase in sCD28 levels just before or at the onset of cancer recurrence. The patient with a complete response entered the stage of progressive disease (PD), where sCD28 levels began to increase from week 46 onwards (Figure 3, upper panel). The patient with a partial response entered the stage of stable disease (SD), where sCD28 levels began to increase at week 35 (Figure 3, lower panel). This indicates that increased and / or increased sCD28 levels may serve as a marker of cancer recurrence after immunotherapy and that this increase may be a predictive marker of impending recurrence.

[0094] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. 1. A method for predicting cancer recurrence in a subject in need thereof, said method comprising measuring soluble CD28 (sCD28) levels in a sample from said subject, wherein an increase in sCD28 levels in said sample from said subject indicates impending cancer recurrence, thereby predicting cancer recurrence in said subject.

2. 10. The method of claim 1, wherein the subject has cancer and is in remission from the cancer, or the subject previously had cancer and is now cancer-free.

3. The method of claim 1 or 2, wherein the subject has undergone or is undergoing immunotherapy.

4. 4. The method of claim 3, wherein the immunotherapy is a PD-1, PD-L1 and / or CD80-based immunotherapy.

5. 3. The method of claim 1 or 2, wherein the increase is compared to sCD28 levels in a sample from the subject prior to relapse.

6. The method of claim 1 or 2, wherein the increase is compared to a predetermined threshold value.

7. 3. The method of claim 1 or 2, wherein the increase is at least a 50% increase.

8. 3. The method of claim 1 or 2, wherein the increase is an increase to sCD28 of at least 1 ng / mL, and optionally the increase is an increase to sCD28 of at least 6 ng / mL.

9. The method of claim 1 or 2, wherein the sample is a blood sample.

10. 3. The method of claim 1 or 2, wherein said measuring comprises measuring at multiple time points, at least one time point is a time point at which the subject is known to be in remission or cancer-free, and at least one time point is a time point at which the subject is at risk of relapse, and optionally remission includes partial and complete responses to therapy.

11. 11. The method of claim 10, wherein the level of sCD28 is less than 5 ng / mL at the time the subject is known to be in remission or cancer-free.

12. 3. The method of claim 1 or 2, wherein the subject prior to the method has a blood sCD28 level of less than 6 ng / mL.

13. 3. The method of claim 1 or 2, wherein the cancer is selected from skin cancer, urothelial cancer, lung cancer, and renal cancer.

14. 14. The method of claim 13, wherein the cancer is selected from melanoma and urothelial cancer.

15. 3. The method of claim 1 or 2, wherein the urgency is within the next 20 weeks.

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