Method of predicting responsiveness to immunotherapy

Abstract

Methods of the disclosure include administration of a cholesterol analog imaging probe and correlation of change in the uptake of exogenous labeled cholesterol analogs as a marker for T cell activation.

Description

METHOD OF PREDICTING RESPONSIVENESS TO IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATION

[0001] The benefit of priority to U.S. Provisional Patent Application No. 63 / 537,286 filed September 8, 2023, is hereby claimed and the disclosure is incorporated herein by reference in its entirety.FIELD

[0002] The disclosure relates to methods of predicting and / or monitoring responsiveness to treatment with an immune checkpoint inhibitor antibody.BACKGROUND

[0003] Immune checkpoint inhibitor antibodies ( IC Is) blocking the PD-1 or CTLA-4 pathways have revolutionized cancer therapy. By preventing signals that turn “off” killing functions of T cells, ICIs can unleash host immunity against cancer cells, producing remarkable survival advantages and even cures for a minority of patients. Unfortunately, the majority of patients do not benefit from ICIs and some patients experience the severe side effects of these drugs. Extending ICIs to breast cancer and other, less immunogenic tumors remains a substantial barrier in cancer immunotherapy. Recently, the anti-PD-1 ICI pembrolizumab received approval for treatment of high-risk, early-stage triple negative breast cancer (TNBC) in July 2021 . TNBC tumors, the least common and most aggressive subtype, typically show more inflammation than other subtypes of breast cancer. An inflamed, immune “hot” environment correlates with better outcomes in TNBC. Unfortunately, tumors in most patients with TNBC are not conducive to immunotherapy based on current metrics for selection and / or do not respond to ICIs. For example, in the KEYNOTE-355 trial, pembrolizumab significantly extended survival in patients categorized as favorable for immunotherapy based on high expression of PD-L1 in tumor cells, lymphocytes, and macrophages in biopsy samples. Despite improved overall survival, anti-PD-1 treatment allowed only 11% more patients to survive through the 4.5-year study endpoint relative to those treated only with chemotherapy. These data highlight shortcomings of existing criteria to predict patients likely to respond to immunotherapy in breast cancer. Creating new, optimally non-invasive methods to improve selection of patients for immunotherapy in TNBC and identify early effects of therapy on T cells will help optimize use of existing drugs and facilitate translation of new immunotherapies. Since only a minority of patients across all malignancies respond to current ICIs, better methods to select patients and monitor therapy likely will improve outcomes across multiple cancers.SUMMARY

[0004] Predicting which patients will benefit from immunotherapy remains a central obstacle to effective use of these drugs for cancer. There are currently three FDA-approved predictive markers for immunotherapy: PD-L1 expression, tumor mutational burden (TMB), and microsatellite instability (MSI). Approved methods rely on tumor biopsies, limiting the ability to repeatedly monitor tumor evolution and response to therapy over time. Several other investigational approaches exist, including gene profiles for T cells and presence of B cells. Listed strategies focus on predicting response to immunotherapy to guide selection of patients. However, none of these methods offer the important capability to detect response to therapy early in treatment.

[0005] Toward the goals of predicting response to ICIs and detecting early effects of therapy, researchers have developed PET imaging probes based on antibodies, antibody fragments such as affibodies or small molecules for the PD-L1 / PD-1 pathway or other immune checkpoint inhibitors. PET imaging studies in mouse models or early clinical studies reveal heterogeneous expression of immune checkpoint proteins in primary tumors and various metastases, emphasizing the value of imaging to detect intra-patient heterogeneity that could affect treatment outcomes. As discussed previously, detecting expression of PD-L1 protein or other immune checkpoint molecules at least by immunostaining has shown limited efficacy to predict patients who will respond to ICIs. Imaging probes to cell surface immune checkpoint inhibitors identifies levels of specific targets, and imaging after starting therapy can detect to what extent an ICI blocks the target molecule. A key limitation is that these probes do not detect to what extent a therapy activates functional T cells, the key effector population of current ICIs.

[0006] [18F]NP-59, is an analog for cholesterol that can be leveraged to detect cholesterol uptake by cells and tissues using positron emission tomography (PET) imaging. [18F]NP-59 overcomes limitations of previous generations of this radiotracer that have been used clinically to detect adrenal cortical tumors, while maintaining sensitivity to detect uptake in tissues and cells, including cells of the immune system. As a cholesterol analog, [18F]NP-59 distributes into the lipid pool and enters lipoproteins with subsequent trafficking into cells through receptors like the low-density lipoprotein receptor (LDLR) and fatty acid transporter, CD36. The fluorine-18 radiolabel is used commonly in radiotracers for PET imaging studies, such as the glucose analog 2-[(18)F] fluoro-2-deoxy-D-glucose to detect glycolytic metabolism in cancer and other diseases. It has advantageously been found that activated and primed T cells in the tumor microenvironment have changed uptake of cholesterol detectable with [18F]NP-59 relative to baseline (e.g., pretreatment) measurements, which can therefore be used as a marker for success of ICI therapy.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 A is a graph showing values ± SEM (symbols) and calculated logistic regression (smooth line) for E0771 tumors (n=5) treated with anti-PD-1 antibody or PBS.

[0008] Figure 1 B is a graph showing E0771 tumor growth for individual mice over time.

[0009] Figure 1C is graphs showing survival curve of mice with E0771 tumors after treatment with anti-PD-1 as analyzed by a Mantel-Cox test.

[0010] Figure 1 D is a graph showing values ± SEM (symbols) and calculated logistic regression (smooth line) for AT3 tumors (n=5) treated with anti-PD-1 antibody or PBS

[0011] Figure 1 E is a graph showing AT3 tumor growth for individual mice over time.

[0012] Figure 1 F is a graph showing a survival curve of mice with AT3 tumors as analyzed by a Mantel-Cox test, demonstrating that anti-PD-1 treatment significantly prolonged survival for mice with E0771 tumors (Figure 1 C), but not AT3.

[0013] Figures 2A and 2B are flow cytometry results of uptake of labeled cholesterol in activated, CD69+ CD8 (A) or CD4 (B) T cells, after isolation of T cells from ICI responsive E0771 or unresponsive AT3 tumors and leaving cells unstimulated (us) or re-stimulated on anti-CD3e coated (stim) dishes for 18 hours in the presence of 3-NBD labeled cholesterol.

[0014] Figures 2C to 2F are graphs illustrating that CD8 (C) and CD4 (D) T cells from E0771 tumors show significantly greater percentages of CD69+ cells with uptake of 3-NBD cholesterol than from ICI non-responsive AT3 tumors. Stimulated CD8 (E) and CD4 (F) T cells from E0771 tumor also exhibited significantly higher mean fluorescence intensity for cholesterol uptake. Data are from 4 experimental replicates with statistical comparisons by Student’s T test.

[0015] Figures 3A and 3B are graphs illustrating (A) CD8+ and (B) CD4+ T cells accumulation of BODIPY-cholesterol in cells from individual tumors from E0771 and AT3 tumors relative to the only / isotype antibody control after injection of C57BL / 6J mice intraperitoneally with cholesterol labeled with red-shifted BODIPY and euthanizing of the animals 24 hours later to collect and dissociate tumors for flow cytometry (n=5 each for E0771 and AT3.

[0016] Figures 3C to 3F are graphs showing that (C) CD8 and (D) CD4 T cells in E0771 tumors showed significantly higher fold change accumulation of fluorescent cholesterol relative to FMO control. (E) CD8 but not (F) CD4 T cells in E0771 tumors also expressed higher levels of PD-1. Differences between means were compared using nonparametric Mann-Whitney tests (n = 5 mice per group) while differences between T cell population percentages were tested using Student’s T test.

[0017] Figure 4A is a graph showing mean values + SEM for total cholesterol and cholesterol esters (n=3 per condition). **, p<0.01 by Student’s T test after activating naive mouse T cells with increasing concentrations of anti-CD3e for 24 hours before quantifying total and esterified cholesterol with a bioluminescence assay.

[0018] Figure 4B is a diagram illustrating the activation of T cells for 24 hours prior to adding different concentrations of the cholesterol radiotracer analog, FNP-59, for one hour before washing and quantifying cell-associated radioactivity by autoradiography with a phosphor imaging screen.

[0019] Figure 4C is a graph showing quantified data for uptake of FNP-59 at different amounts of administered radiotracer. All experiments were performed in triplicate, using comparison of fits with the extra sum-of-squares F test used to compare and test each data set and EC50 against the null hypothesis.

[0020] Figure 4D is a graph showing quantified data of T cells isolated from healthy human PBMC that were activated with human anti-CD3e and treated and analyzed as described for mouse T cells.

[0021] Figure 4E is a plot using annotated T cell clusters from the Immunological Genome Project’s (Immgen) Immune Cell Atlas scRNA-seq data sets on healthy human lamina propria, genes that involved in cholesterol uptake, retention, and trafficking were analyzed.

[0022] Figure 4F is a panel displaying fold change in selected genes related to cholesterol metabolism between the ‘highly activated T cell’ cluster as compared with all other T cells.

[0023] Figure 5A is a graph showing uptake of FNP-59 per microgram of spleen tissue measured by scintigraphy. Tumors analysis was performed after treatment with anti-PD-1 antibody or PBS for 4 days after tumors reached 70 mm2and administration of 100 pCi of FNP-59 after the treatment. Symbols show individual mice with annotations for mean values and standard deviations.

[0024] Figure 5B is a graph showing accumulation of FNP-59 normalized to total cell protein with BCA assay of CD4 and CD8 tumor infiltrating lymphocytes (TIL) isolated by positive selection with immunomagnetic beads and determined. Representative data from 2 experimental replicates with statistical comparisons by Student’s T-test.

[0025] Figure 6A is an image showing re-analysis of single-cell RNA sequencing data with Cellenics software displays Louvain clusters of annotated T cell states: T memory / resting, highly activated, T effector / transitional state, and T effector / dysfunctional.

[0026] Figure 6B is a plot shows proportions of the absolute counts across for various T cell subsets across TNBC patients (n =8429 cells from 9 tumors).

[0027] Figure 6C is a graph showing clustered averaged gene expression data reveal upregulation of relevant genes involved in uptake and intracellular trafficking of cholesterol in cycling T cells.

[0028] Figures 6D to 61 are violin plots of normalized expression of specific genes: D) SREBF2 (positive regulation of cholesterol uptake and synthesis), (E) LDLR (cholesterol uptake), (F) STARD3NL and (G)LAMTOR (endosomal transport), (H)COMMD1 (LDLR recycling), (l)ANXA2 (cholesterol distribution to the plasma membrane). I Indicates P < 10-20between “cycling” cluster and other T cell clusters.

[0029] Figure 7 is a table showing increased expression of genes related to cholesterol uptake in cycling T cell populations in patients with triple-negative breast cancer. Major T cell populations from existing single-cell RNA sequencing data were re-analyzed using Cellenics software by Biomage (see Figure 6). With Reactome analysis software interface, the “cycling” cluster were compared to all other T cells using Enrichr for pathways ontologically related to cholesterol uptake. Filter cutoffs used P < 0.05 and log FC > 0.1 .

[0030] Figure 8 is a chart showing adjusted P-values and logFC values of specific genes expressed when comparing Louvain clusters from Figure 6. Rest / mem is the annotated resting / memory T cell population, Transit / eff is the transitional / effector T cell population, and Dysfunct is the dysfunctional T cell population.

[0031] Figure 9 shows standard curve and luminescence image for luminescent cholesterol assay shown in figure 4A. Plate image after I VIS imaging. Anti-CD3 concentrations were plated decreasing in rows top to bottom as labeled and in duplicate with esterase added on the right (for total cholesterol).

[0032] Figure 10 is plate images after phosphor screen processing from Figure 4C. FNP- 59 concentrations were plated increasing left to right in triplicate. The six concentrations represented by light gray to black color boxes were 0.05, 0.1 , 0.25, 0.5, 0.75, 1 pCi treatments over 1 hour.DETAILED DESCRIPTION

[0033] Activated T cells are one of the hallmarks of an inflamed, effective anti-tumor immune response. Previous studies suggest a link between activation of T cells and cholesterol. T cells increase cholesterol through de novo synthesis from acetyl CoA or uptake from lipoproteins through receptor-mediated endocytosis of low-density lipoprotein (LDL) particles from the bloodstream. Cholesterol is an essential component of cellularmembranes, where this lipid modulates membrane fluidity, permeability, and receptor mediated signaling. Upon activation, T-cell receptor (TCR) nanoclusters form in cholesterol- rich membrane domains to elicit strong TCR signaling. Proliferating T cells and other cell types require increased amounts of cholesterol to replicate cell membranes, utilizing cholesterol stored as cholesterol esters in intracellular lipid droplets. Despite requirements for cholesterol in T cell activation and proliferation, increased cholesterol uptake by tumor infiltrating T cells has been linked to an “exhausted” T cell phenotype. However, exhausted T cells indicate a history of active T cell populations, and both populations of T cells correlate with success of ICIs in TNBC. Therefore, cholesterol uptake by tumor infiltrating lymphocytes could indicate a “hot” tumor environment and predict the efficacy of immunotherapy.

[0034] Tumors can generally present as a 1 ) immune desert phenotype lacking immune cells and immune signaling, 2) an immune excluded phenotype in which immune cells encounter mechanical and cell signaling barriers preventing tumor infiltration, and 3) immune infiltrated or inflamed phenotype. TNBC in humans is heterogeneous, but presents with an inflamed microenvironment more frequently than other subtypes of breast cancer. Although generally considered more inflamed, TNBC can exist anywhere on this continuum of immune phenotypes and exhibits substantial intra- and intertumoral heterogeneity. Tumors with high infiltration of lymphocytes such as NK cells, cytotoxic T cells, and B cells correlates with more favorable prognosis. The key publication from Hu et. al, 2021 , Immune Classification and Immune Landscape Analysis of Triple-Negative Breast Cancer. Frontiers in Genetics, identified three major immune subtypes. Only the subtype with enrichment of lymphocytes and immune checkpoint ligands showed favorable outcome. However, even the most favorable subtype of TNBC demonstrated heterogeneity in TIL function and overall efficacy (through modulation of key genes). Clinical trials and treatment with immunotherapy rely on a combined positive score (CPS) to subclassify patients and predict response. The CPS system analyzes numbers of PD-L1 (or other marker) positive tumor cells, lymphocytes, and macrophages out of total number of viable cancer cells in histologic sections from biopsies. While used widely, the CPS may vary based on selection of different antibodies for immunostaining, and CPS cut-offs have only modest success for predicting response to ICIs. Therefore, identification of more informative and potentially non-invasive biomarkers for efficacy of ICIs remains a highly active area of translational research.

[0035] It has advantageously been found that the uptake of cholesterol analog imaging probes can be correlated to a predicted responsiveness to immunotherapy treatment and / or be useful in monitoring the effectiveness of such therapy. It has been observed that a change in uptake amount, either increased or decreased, can be indicative of responsiveness to ICIs. The change in uptake of the exogenously administered cholesterolimaging probe is representative of a change in the environment of the cancerous region, which can be correlative to responsiveness to treatment. In particular, the environment of a cancerous region generally includes at macrophages, T-cells and cancer cells as the major source for uptake in cholesterol. Imaging of the uptake of the cholesterol analog imaging probe provides a net uptake of these sources in the region being imaged. For cancers responsive to treatment, an increase in uptake of the cholesterol imaging probe in the cancerous region can be seen where the environment being scanned is rich in T-cells. Activation of the T-cells resulting from responsiveness to the ICI can result in an increase in the uptake of the cholesterol imaging probe. If the environment of the cancerous region being scanned, however, is rich in macrophages, responsiveness to the ICI may shift the state of the macrophage from M2, which is a state of higher cholesterol use, to the state of M1 , in which they are active and utilize sugar, with reduced cholesterol uptake. This change in state, corresponding to reduced cholesterol analog imaging probe uptake is also believed to be indicative of responsiveness to ICIs.

[0036] The change in uptake of the cholesterol probe can be determined as a change in the standardized uptake value, for example, Standardized uptake value (SUV) dimensionless ratio, which is well known as a clinical metric in nuclear medicine. It has been observed that changes in the standardized uptake value of the administered cholesterol analog probe, as determined by imaging the cancerous region, can be as much as 3x different after administration of the immune checkpoint inhibitor as compared to the uptake prior to treatment. For example, a ratio of the standardized uptake value after treatment to the standardized uptake value before treatment can be 3:1 to 1 :3. Changes less than 3x or ratios less than 3:1 to 1 :3 are also contemplated here and the degree of change can vary with the treatment and tumor type while still being indicative of responsiveness to the immunotherapy. A statistically significant changes in uptake between two images can be recognized based on knowledge in the art regarding uptake of imaging probes and imaging modalities. For example, it is known the art that a change, increase or decrease, in standardized uptake value of 0.5 to 1 is indicative of a statistically significant change in the uptake beyond any inherent variation or standard of error of the imaging method, such as, for example PET imaging.

[0037] The cancerous region can be, for example, a tumor. Methods of the disclosure can be useful in predicting ICI-responsive tumors. Changes in uptake of the cholesterol analog probe before and after treatment are indicative of changes in the tumor environment and responsiveness to the therapy administered.

[0038] Methods of the disclosure can include contacting cells from a tumor and / or a tumor biopsy sample with a cholesterol analog imaging probe and measuring an uptake of theimaging probe by the cells. For example, detectable uptake of the imaging probe can be predictive of responsiveness of treatment with an ICI, whereas nondetectable uptake is predictive of non-responsiveness to ICI treatment. For example, increased uptake of the imaging probe, for example, as compared to a baseline, can be used to predict the tumor will be responsive to ICI treatment. The baseline can be, for example, healthy non-tumor cells.

[0039] Methods of the disclosure can include evaluating an effectiveness of treatment by contacting cells from a tumor and / or a tumor biopsy sample with a cholesterol analog imaging probe and measuring an uptake of the imaging probe by the cells prior to treatment, treating the subject or contacting the sample with an ICI for a treatment duration, and then contacting the cells from the tumor and / or a tumor biopsy sample after treatment for a treatment duration with a cholesterol analog imaging probe and measuring an uptake of the imaging probe by the cells after treatment. A change in the amount of uptake, either increased or decreased, can be indicative of responsiveness to treatment, while maintenance of substantially the same uptake level can be indicative of non-responsiveness to the treatment.

[0040] Methods of the disclosure can include administering to a cancer patient a cholesterol analog imaging probe and measuring an uptake of the imaging probe to predict a responsiveness of the patient to treatment with an ICI. The method can include administering the cholesterol analog imaging probe to the patient after treatment for a treatment duration and measuring an uptake of the imaging probe. A change in uptake, either increased or decreased, relative to a baseline of a cohort patients with non-responsive tumors of the same cancer type can be indicative of responsiveness to the treatment.

[0041] Methods of the disclosure can include intravenously administering the imaging probe. Methods of the disclosure can include site-directed administration of the imaging probe to a tumor site in vivo. In the methods of the disclosure, increased uptake of the cholesterol analog imaging probe can be correlative to predicted responsiveness to treatment with ICI. Increased uptake can be measured as compared to a baseline. The baseline can be, for example, uptake of the imaging probe by healthy individuals.

[0042] Methods of the disclosure can include monitoring responsiveness of a patient to immunotherapy. Such methods can include administering a cholesterol analog imaging probe to a patient prior to treatment with the immunotherapy and measuring an uptake of the cholesterol analog imaging probe and then administration of the cholesterol analog imaging probe after treatment and measuring an uptake of the cholesterol analog imaging probe. A comparison of the uptake before and after immunotherapy treatment can be indicative of responsiveness of the disease to the immunotherapy treatment. An increase or decrease inuptake of the cholesterol analog imaging probe has been advantageously found to correlate to responsiveness to the immunotherapy.

[0043] In any of the methods of the disclosure, the method can include or further include ex vivo analysis of this cholesterol analog imaging probe to measure redistribution of cholesterol in a tumor biopsy sample.

[0044] In methods of the disclosure, the cholesterol analog imaging probe can be used before biopsy or surgery, followed by isolation of T cells and ex vivo imaging of accumulation. Alternatively, since early responses to ICI therapy include activation of peripheral blood CD8 T cells enriched for anti-tumor T cell clones, changes in accumulation of cholesterol analog imaging probe in blood samples collected before and after treatment could monitor efficacy of an immunotherapy.

[0045] In any of the methods of the disclosure, measuring an uptake of the cholesterol analog imaging probe can be done using any known medical imaging technique. For example, medical imaging techniques, such as Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), can be used. These techniques utilize radionuclide containing contrast agents, detected by complex detectors that are combined with computational techniques to develop three-dimensional images of internal organs and features. Generally speaking, PET provides imaging that is significantly higher resolution than SPECT (5-7 mm compared to 12-15 mm, respectively). Additionally, PET has recently been adapted to enable quantification of medical imaging, which has not been accomplished with SPECT. The cholesterol analog imaging probe used in the methods of the disclosure is described in detail below and can include a radionuclide to allow for quantification using medical imaging technologies. For example, if imaging with SPECT CT, the cholesterol analog imaging probe can include an iodine label. For example,18F or124l can be used as the label in the cholesterol analog imaging probe for PET, PET Ct or PET MRI imaging.123l,125l or1311 can be used as the label in the cholesterol analog imaging probe for SPECT or SPECT CT imaging. Selection of a suitable label for inclusion on the cholesterol analog can be selected based on the imaging technique to be used. Other isotopes include76Br and77Br, which could be utilized for PET or SPECT imaging respectively.

[0046] In any of the methods of the disclosure the patient can have one or more disease including cancer. For example, the disease can be a disease that is responsive or suspected to be responsive to treatment with an immune checkpoint inhibitor. For example, the disease can be a disease that is responsible of suspected to be responsive to treatment with one or more PD1 , PDL1 , CTLA-4, LAG-3, checkpoint inhibitor. For example, the cancer canbe lung cancer, cervical cancer, colon cancer, bladder cancer, breast cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, skin cancer, stomach cancer, rectal cancer, or solid tumor. For example, the cancer can be melanoma, for example the melanoma can be of the head and / or neck, and / or squamous cell cancer. The cancer can be, for example, triple negative breast cancer. For example, the disease can be an autoimmune diseases or disorder. For example, the autoimmune disorder or diseases can be rheumatoid arthritis. For example, the disease can be a chronic infection.

[0047] The cholesterol analog for use in the methods of the disclosure can be a halogenated cholesterol analogue as described in U.S. Patent Application Publication No. 2021 / 0355154, the disclosure of which is incorporated herein by reference. The cholesterol analogs for use in the methods of the disclosure can be made as described in the ‘154 publication.

[0048] For example, the cholesterol analog can be compound Formula (I):wherein the substituents are described in detail below. For example, the methods of the disclosure can include administration of the cholesterol analog [18F]NP-59, which has the structure

[0049] Uptake of cholesterol as a novel marker for efficacy of immunotherapy in mouse models of TNBC was evaluated. Increasing levels of TCR activation were observed to be correlated with higher uptake of cholesterol in T cells. Methods of the disclosure capitalize on this observation and leverage cholesterol analog imaging probes as a radiotracer for imaging cholesterol trafficking and uptake as a marker for efficacy of ICI . In the examples herein, [18F]NP-59 was used as the cholesterol analog imaging probe. For tumor studies,two mouse models of TNBC, EO771 and AT3, were utilized that are responsive or not responsive to immunotherapy, respectively. Ex vivo restimulated and in vivo T cells from ICI responsive breast tumors in mice accumulated significantly more cholesterol than T cells from ICI non-responsive tumors. Remarkably, uptake of [18F]NP-59 was detected within three hours of injection in tumor-bearing mice. T cells from tumors responsive to ICI therapy showed higher uptake of [18F]NP-59 under basal conditions and following anti-PD1 therapy. In human patients with TNBC, populations of “activated T cells” exhibited gene signatures of enhanced cholesterol uptake as compared with resting, effector, and exhausted T cell states. This gene signature included upregulation of key genes, including ANXA2, a multifaceted protein that works with CD36 for lipid scavenging. Additionally, it was found that upregulation of the master transcriptional regulator of cholesterol metabolism, SREBF2.

[0050] In the examples of the methods of the disclosure, discussed herein, greater success of ICI therapy in breast tumors wherein T cells accumulated greater cholesterol from the extracellular environment. A recent study comprehensively mapped cholesterol metabolism and disposition in tumor environments. Intratumoral T cells showed cholesterol deficiency, which impaired proliferation and triggered autophagy-mediated cell death. Across all cells in a tumor, tumor cells and myeloid lineage cells contained highest amounts of cholesterol with lower overall levels in T cells. I ntriguingly, suppressing cholesterol efflux pathways by targeting the LXRp transcription factor enhanced anti-tumor functions of T cells. These data supported the findings that activated T cells accumulate more cholesterol and point to the benefits of the methods of the disclosure in providing a mechanism to track disposition of cholesterol in tumor environments.

[0051] It has been found that uptake of cholesterol marks activation of T cells in mouse TNBC tumors responsive to ICI therapy, which has surprisingly allowed for distinguishing T cells from ICI-responsive and non-responsive tumors with a cholesterol analog imaging probe, such as [18F]NP-59. Ex vivo restimulation of T cells recovered from ICI responsive tumors in mice exhibited greater cholesterol uptake compared to non-responsive tumors. In vivo, it was discovered that T cells from responsive tumors accumulated significantly more cholesterol than non-responsive tumors, the latter of which exhibited almost undetectable uptake. Lastly, data mining studies of human TNBC showed upregulation of genes-related to the uptake and trafficking of cholesterol in populations of activated T cells. These data demonstrate that uptake of cholesterol as measured with a cholesterol analog imaging probe demarks proliferating T cell populations and correlates with success of immunotherapy in pre-clinical mouse models of TNBC.Cholesterol Analog Imaging Probes

[0052] The cholesterol analog imaging probe used in the methods of the invention can be any known cholesterol analog that includes a label that is a halogen or radiohalogen.Selection of the label can depend on the imaging method to be used as detailed above. The halogen can be, for example, I, F, CL, or Br. The label can be, for example, a radioisotope of any of I, Cl, F or Br.

[0053] For example, the cholesterol analogs used in the method of the disclosure can a structure of Formula (I), whereinR1is OH or OP;R2, when present, is OH or X;R3is H, OH, X, CH2-X, or CH2-LG;R4, when present, is C1-6 alkyl, C1-6 alkylene-X, or C1-6 alkylene-LG;X is a halogen;P is an alcohol protecting group;LG is a leaving group; each of bond A and bond B is a single or a double bond and only one of bond A and bond B can be a double bond; and with the proviso that at least one X is present as the label.

[0054] In some cases, the compound of formula I can be such that at least one X or LG is present; and if LG is present, R1is OP. In some cases, the compound of formula I can be such that if one R2and R3is F and the other OH, then the F is18F.

[0055] In some cases, the compound formula I can optionally be

[0056] As disclosed herein, X is a halogen. In certain embodiments, X is F, Cl, I or Br.

[0057] In embodiments, X can be a radioisotope. As used herein, a “radioisotope” refers to an unstable, radioactive isotope that emits excess energy in the form of one or more of a, P, and y radiation. Examples of common radioisotopes of halogens include, for example,37CI,18F,77Br,124l, and1311. Furthermore, as used herein, a “hot” compound refers to any compound including a radioisotope, whereas a “cold” compound refers to any compound including a stable, non-radioactive isotope. Accordingly, the terms “hot” and “radiolabeled” can be used interchangeably, while the terms “cold” and “non-radiolabeled” can be used interchangeably.

[0058] In some cases where X is F, X is specifically18F. In some cases where X is I, X is specifically123l,124l,125l or1311. In some cases where X is Br, X is specifically76Br or77Br

[0059] In certain aspects, R1is OH. In other aspects, R1is OP. In various cases, P is pivaloyl, acetoxy, THP, or MOM. In embodiments, P is THP or MOM.

[0060] In certain aspects, R2is X. In other aspects, R2is OH.

[0061] In various aspects, R3is X or CH2-X. In some embodiments, R3is CH2-LG. In embodiments, LG is tosyl, a halogen, mesyl, or triflate. In some embodiments, LG is tosyl or mesyl.

[0062] In some aspects, R4is Ci-6alkylene-X.

[0063] In various cases, A is a double bond. In other cases, B is a double bond. In some cases, each of A and B is a single bond.

[0064] In some embodiments, the compound has a structure of Formula (IA):wherein R3is C1-6 alkylene-X or C1-6 alkylene-LG. In some aspects, R1is OP and R3is CH2- LG. In some aspects, P is acetoxy and LG is OTs. In other cases, P is MOM or THP and LG is OTs or OMs. In some cases, R3is CH2-OTS or CH2-OMS. In some embodiments, R1is OP and R3is CH2-X. In some cases, P is pivaloyl and LG is OMs.

[0065] In some embodiments, the compound has a structure of Formula (IB):wherein R4is C1-6 alkylene-X or C1-6 alkylene-LG. In some aspects, R1is OP and R4is C1-6 alkylene-LG. In some cases, P is acetoxy and LG is OTs. In other cases, P is MOM or THP and LG is OTs or OMs. In some embodiments, R4is CH2-OTS or CH2-OMS. In some cases, R1is OH and R4is C1-6 alkylene-X. In some cases, R4is CH2-X.

[0066] In some embodiments, the compound has a structure of formula (IC)wherein one of R2and R3is OH and the other is X, and R4is C1-6 alkylene. In some aspects, R4is methyl. In some cases, R2is X and R3is OH. In other embodiments, R2is OH and R3is X.

[0067] In some embodiments, the disclosure provides compounds having a structure

[0068] In some aspects, the compound has a structure selected from:Chemical Definitions

[0070] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups. The term Cn means the alkyl group has “n” carbon atoms. For example, C4 alkyl refers to an alkyl group that has 4 carbon atoms. C1-6alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms), as well as all subgroups (e.g., 2-6, 1-5, 3-6, 1 , 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n -propyl, isopropyl, n- butyl, sec-butyl (2-methylpropyl), and t-butyl (1 ,1 -dimethylethyl). Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0071] As used herein, the term "alkylene" refers to a bivalent saturated aliphatic radical. The term Cn means the alkylene group has "n" carbon atoms. For example, C1 -6 alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for "alkyl" groups.

[0072] As used herein, the term “epoxy” or “epoxide” refers to a three-membered ring whose backbone comprises two carbon atoms and an oxygen atom.

[0073] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine. In some cases, the halo is a radioactive halogen. Examples of radioactive halogens include, but are not limited to, fluorine-18, chlorine-37, bromine-77, and iodine-124, iodine- 131 .

[0074] As used herein, the term “leaving group” refers to any atom ar moiety that is capable of being displaced by another atom or moiety in a chemical reaction. Examples of suitable leaving groups include, but are not limited to, a dialkyl ether, triflate, tosyl, mesyl, and a halogen.

[0075] As used herein, the term “alcohol protecting group” refers to a group introduced into a molecule by chemical modification of an alcohol (i.e. hydroxyl) group in order to obtain chemoselectivity in a subsequent chemical reaction and to prevent modification of the alcohol group under certain conditions. Examples of suitable alcohol protecting groups include, but are not limited to, methyl, t-butyloxycarbonyl (Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1 -methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4- methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1 ,4-dioxan-2-yl, tetrahydrofuranyl,tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2- yl, 1 -ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1 -methyl-1 -methoxyethyl, 1 -methyl-1 - benzyloxyethyl, 1 -methyl-1 -benzyloxy-2-fluoroethyl, 2,2,2-trich loroethyl , 2-trimethylsilylethyl , 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl- 2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1- yl)bis(4',4"-dimethoxyphenyl)methyl, 1 ,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1 ,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldimethylsilyl (TBDMS or TBS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p- phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p- methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4- methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4- (1 ,1 ,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1 ,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o- (methoxyacyl)benzoate, .alpha.-naphthoate, nitrate, alkyl N,N,N,N'- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesyl), benzylsulfonate, andtosyl (Ts). In some cases, the alcohol protecting group is methoxymethyl ether (MOM), tetrahydropyranyl ether (THP), t-butyl ether, allyl ether, benzyl ether, Fbutyldimethylsilyl ether (TBDMS), t-butyldiphenylsilyl ether (TBDPS), acetoxy, pivalic acid ester, or benzoic acid ester. In some cases, the alcohol protecting group is MOM or THP.

[0076] The manner of administration of the cholesterol analog imaging probe is not particularly limited. For example, cholesterol analog imaging probe can be administered intravenously or orally. The manner of administration and dose thereof would be within the purview of the doctor, nurse, or radiologist trained to administer these compounds.

[0077] In embodiments, the imaging modality can be selected from positron emission tomography (PET), positron emission tomography / computed tomography (PET / CT), positron emission tomography / magnetic resonance imaging (PET / MRI), planar gamma camera imaging, single-photon emission computerized tomography (SPECT), and / or single-photon emission computerized tomography / computed tomography (SPECT / CT).

[0078] Generally, it is envisaged that cholesterol analog imaging probe include a radioisotope when the subject is subjected to the imaging modality. However, in particular embodiments, the compound can include a non-radiolabeled compound, that is, a compound including, for example,19F, and still remain suitable for imaging. For example, PET / MRI can be used to image cold compounds, such as those including19F or127l.

[0079] The subject can be subjected to the imaging modality at a point in time ranging from about 0.5 hours to 7 days after of the compound. The time at which the subject is subjected to the imaging modality is dependent on the isotope of the halogen used in the cholesterol analogue. For example, due to the short half-life of18F, when the compound is radiofluorinated, the subject can be subjected to the imaging modality at a point in time ranging from about 0.5 hours to 18 hours, about 12 hours to 18 hours, 0.5 hours to about 5 hours, about 0.6 hours to about 4.5 hours, about 0.7 hours to about 4 hours, about 0.8 hours to about 3.5 hours, about 0.9 hours to about 3 hours, or about 1 hour to about 2 hours, for example at about 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8 ,9, 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18 hours after administration of the compound. Due to the half-life of124l, for example, having a ti / 2 = 4.2 days, when the compound is radioiodonated, the subject can be subjected to the imaging modality at a point in time ranging from about 0.5 hours to about 7 days, from about 5 hours to about 5 days, from about 12 hours to about 3 days, or from about 1 day to about 2 days, for example at about 0.5 hours, about 1 hour, about 2 hours, about 5 hours, about 7 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the compound.EXAMPLESMethods and MaterialsCell lines

[0080] Mouse EO771 (ATCC) and AT3 (EMD Millipore) breast cancer cells and C57BL / 6J mouse mammary fibroblasts (gift of Harold Moses, Vanderbilt University) were cultured in DMEM base media (Gibco, #11965) supplemented with 10% FBS, 1% GlutaMax (ThermoFisher Scientific, #35050061 ), and 1% penicillin-streptomycin (Gibco, #15140148). All cells were maintained in a humidified 37°C incubator with 5% CO2.Mouse tumor models

[0081] The University of Michigan Institutional Animal Care and Use Committee approved all animal procedures under protocol PR000010534. 5x105EO771 (ATCC) or AT3 (EMD Millipore) mouse mammary tumor cell lines and 1x105mouse mammary fibroblasts orthotopically were implanted into 4thinguinal mammary fat pads of 6-8-week-old female C57BL / 6J female mice (The Jackson Laboratory). Mice were housed in specific pathogen- free housing facilities maintained by the Unit for Laboratory Animal Medicine at the University of Michigan. Three days after implanting cells, mice were randomly assigned to treatment with anti-PD-1 antibody (10 mg / kg intraperitoneal) (BioXCell, #CD279) or PBS administered every three days for four total doses. Growth of orthotopic tumors were measured with calipers twice per week, quantifying tumor size for each mouse as mm2by multiplying orthogonal length x width measurements. Mice were euthanized for a predetermined humane endpoint of tumor size, which defined time of censoring for survival analyses.T cell assay preparation, reagents, and antibodies

[0082] T cells from the spleens of wild-type C57BL / 6J mice were purified by processing tissue through a 70-micron filter, lysing red blood cells with ACK buffer (vendor and number), and sorting cells using the Pan T Cell Isolation Kit II from Miltenyi (#130-095-130). For ex vivo activation of T cells, 96 well plates were coated with indicated amounts of anti-CD3e antibody supplemented with soluble anti-CD28 at 2-times the amount of anti-CD3e and 5U / mL hL IL-2 (Biolegend). For activation, specific antibodies included anti-mouse CD3E (145-2C11 ) and CD28 (37.51 ) or anti-human CD3 antibody (OKT3) and anti-human CD28 (CD28.2) (antibodies from Biolegend). For flow cytometry experiments, antibodies included anti-mouse CD45(S18009D), TCR chain (H57-597), CD3 (17A2), CD8a (53-6.7), CD4 (RM4-5), CD69 (H1.2F3), and CD279 (29F.1A12) (Biolegend). Cholesterol probes includedCholesteryl BODIPY 542 / 563 C11 (Invitrogen, #C12680) and 3-dodecanoyl-NBD Cholesterol (Cayman Chemicals, #13220) for in vivo and in vitro experiments respectively.Synthesis of [18F]FNP-59

[0083] The synthesis of [18F]FNP-59 was accomplished as previously described for clinical research studies and as described in U.S. Patent Application Publication No. 2021 / 0355154. [18F]Fluoride was produced via the18O(p,n)18F nuclear reaction with a GE PETtrace cyclotron equipped with a high-yield fluorine-18 target. [18F]Fluoride was delivered in a bolus of [18O]H2O to the synthesis module and trapped on a QMA-Light sep-pak cartridge to remove [18O]H2O. [18F]Fluoride was then eluted into the reaction vessel with potassium carbonate. Acetonitrile with Kyrptofix 2.2.2 was added to the reaction vessel, and the [18F]f luoride was azeotropically dried by heating the reaction vessel to 100 °C and drawing full vacuum. After this time, the reaction vessel was subjected to both an argon stream and a simultaneous vacuum draw at 100 °C. The solution of precursor in DMSO was added to the dried [18F]fluoride, and it was heated at 120 °C with stirring for 20 min. Ester group was removed by treatment with KOH and heating at 110 °C for 25.18[F]FNP-59 was isolated via reverse phase chromatography and reformulated into a solution of saline, ethanol, and Tween-80.In vitro / ln vivo uptake of [18F]NP-59

[0084] For experiments measuring T cell accumulation of [18F]NP-59 in vitro, 96 well plates were washed with activated cells once before adding radiotracer at indicated concentrations (n = 3 wells per concentration). The plates were incubated for 1 hour and then washed twice with RPMI medium (Gibco, #11875903) containing 0.1% serum before quantifying retained radioactivity in each well. All radioactivity measurements were performed for experimental wells and a standard curve with a phosphor imaging screen (GE Typhoon FLA 7000). To quantify radioactivity by densitometry image analysis, ImageQuant Molecular Dynamics) was used and plotted data with Prism GraphPad version 9 (San Diego, CA, USA).

[0085] For mouse experiments, 100 pCi of18[F]NP-59 was intravenously injected three hours before imaging selected mice or euthanizing mice to recover spleen and tumorinfiltrating T cells. All other mice were euthanized three hours after injection. To recover T cells for analysis, tumors were dissociated in 5mL of tumor dissociation buffer containing RPMI base media, 5% FBS, 300U / mL Collagenase I, 150U / mL Collagenase IV and,200ug / mL of DNasel (VENDORS), using the TDK1 program on the gentleMACS Octo Dissociator (Miltenyi, Waltham, MA, USA). After 30 minutes of incubation, tumor tissue was passed through a 70-micron filter and isolated T cells using CD4 / CD8 (TIL) MicroBeads and cell sorting magnets from Miltenyi. Recovered T cells were placed into 1 .5mL tubes and measured decay-corrected radioactive counts with a Perkin Elmer Gamma Counter. The accumulation of [18F]NP-59 was quantified in excised whole spleens. Accumulated radiotracer activity was normalized to total protein via BCA assay (Pierce assay) for T cells and whole organ weight for the spleen, respectively.In vitro / In vivo fluorescent cholesterol uptake and flow / spectral cytometry

[0086] For in vitro experiments measuring uptake of fluorescent cholesterol analogs during early T cell activation, 1 ug / mL 3-NBD cholesterol was added to 96 well plates with T cell activation conditions for 24 hours. T cells were washed once with PBS and prior to staining in the plate using antibodies against T cell identification markers (TCRp, CD4, and CD8) and activation marker CD69. Cells were analyzed on the same day using a Bio-Rad Ze5 Analyzer (Bio-Rad, Hercules, CA, USA). Single color controls enabled compensation and spectral unmixing of samples, and fluorescence minus one (FMO) control ensured no autofluorescence or incidental FRET with emission from BODIPY. The following gating strategy was used: 1) FSC / SSC to exclude debris; 2) single cells defined by FSC-H / FSC-A gated on the linear population; 3) live cell populations using live-dead exclusion dye (Zombie Aqua, #423101 , Bio-Rad); 4) CD45+ to include only white blood cells; 5) TCRp constant chain-positive to include all T cells; and 6) CD4 or CD8 gates and finally analytic antibodies or probes including CD69, PD-1 , or cholesterol.

[0087] To analyze total T cell cholesterol content, the Cholesterol / Cholesterol Ester-Glo™ Assay from Promega was used. The experiments were setup in a 96 well plate with 105cells per well. Cells were stimulated with plate bound anti-CD3 using 0.01 , 0.1 , 1 , and 10 pg / mL and an additional group was unstimulated. Soluble anti-CD28 was provided to all groups at three times the amount of anti-CD3 for each group and IL-2 was excluded to prevent the impact of rapid growth stimulation and dissociate the effect from added growth factor / high affinity IL-2 receptor expression. After 24 hours, samples were diluted in Cholesterol Lysis Solution and the manufacturer’s procedure was followed with 1 :8 and 1 :20 dilutions of sample. This protocol measured total cholesterol with samples assayed plus esterase in Cholesterol Detection Reagent and free cholesterol with samples assayed minus esterase in Cholesterol Detection Reagent. Cholesterol ester levels were calculated as the difference between total and free cholesterol. Luminescence was recorded on an I VIS Lumina LT series III (Perkin Elmer).

[0088] For in vivo experiments, mice were injected intraperitoneally with 150 pg of BODIPY labeled cholesterol in 50% DMSO + PBS (added directly before injection) or vehicle only. After 1 day, mice were euthanized; tumors were removed; and each tumor was weighed. Tumors were dissociated as described for radiotracer studies and used 106cells for staining for flow cytometry.Data mining of scRNA-seq data

[0089] To reanalyze data from Wu SZ et al. 2021 , sequencing files from the Single Cell Portal hosted by the Broad institute were downloaded at https: / / singlecell.broadinstitute.org / single_cell / study / SCP1039 / a-single-cell-and-spatially- resolved-atlas-of-human-breast-cancers#study-download(brca_mini_atlas_raw_unfiltered.zip). The dataset was analyzed with Cellenics software (Biomage). Analysis included reprocessing the data by classifier filter, mitochondrial content filter, GenesVslIMI filter, doublet filter, data integration with Seurat v4, and LIMAP embedding. Data processing settings can be found in the supplemental file “data processing settings.txt with data found in supplemental file “processed_matrix.rds”. Cellenics web user interface provided further options for data visualization including UMAP, cluster cell counts, DOT plots and individual gene violin plots.

[0090] To analyze data from, Martin, et. al. 2019, the web user interface from The Broad Institute at, https: / / singlecell.broadinstitute.org / single_cell / study / SCP359 / ica-ileum-lamina- propria-immunocytes-sinai?scpbr=immune-cell-atlas#study-visualize was used as directed from the Immgen database at https: / / www.immgen.org / Databrowser19 / HumanExpressionData.html. The data was retrieved from the web user interface and compared the selected genes between activated T cells and other effector populations. Data can be retrieved and replicated through the above link.Statistics

[0091] All data processing was performed in Prism Graph Pad V9. Student’s t test was used to compare statistical significance between groups of normally distributed samples and mean fluorescence intensity, or relative means were analyzed using nonparametric Mann- Whitney tests. Asterisks (ns = p > 0.05; *p < 0.05, **p < 0.01 ,***p < 0.001 , ****p < 0.0001 ) indicate the degree and magnitude of significance between two groups. Kaplan-Meier plots were created and analyzed differences in survival using the Gehan-Breslow-Wilcoxon test. In assessing statistical differences of tumor burden over time between in vivo groups, nonlinear regression was used for logistical growth to determine significant differences between growth curves. Similarly, for in vitro studies using18[F]NP-59, non-linear regression and testsbetween data set parameters were used to assess significance. A p-value cutoff of p < 10-20defined significant differences in expression of specific genes between Louvain clusters.Example 1T cells from ICI responsive tumors showed greater cholesterol uptake

[0092] To study cholesterol uptake and the immunotherapy response in tumor infiltrating T cells, cell lines that model TNBC, but exhibit different responses to immunotherapy were used. EO771 cells, one of the only mammary tumor cell lines derived from spontaneous tumorigenesis in C57BL / 6J mice, responds to immunotherapy and has been used to test effects of therapeutic agents to improve immunogenicity. In contrast, AT3 cells are derived from transgenic MMTV-PyMT mice and respond poorly to immunotherapies, including anti- PD-1 , despite the presence of intertumoral T cells. To directly compare effects of anti-PD-1 antibody therapy on tumor growth, either EO771 or AT3 breast cancer cells with syngeneic immortalized mouse mammary fibroblasts were orthotopically implanted. Three days after implanting cells, mice were randomly assigned to treatment with anti-PD-1 antibody therapy or vehicle. Mice received a total of four doses administered every 3 days. Tumor size was quantified by caliper measurements and monitored survival until mice reached humane endpoints for tumor burden. Mice with EO771 tumors showed remarkable reductions in tumor growth (Figure 1 A & 1 B) and significantly increased overall survival in response to anti-PD-1 therapy (Figure 1C). By comparison, anti-PD-1 treatment elicited no change in tumor growth (Figure 1 D & 1 E) or survival over control in mice with AT3 tumors (Figure 1 F). These data validate responsive and treatment refractory mouse models of immunotherapy in TNBC, providing contrasting systems to investigate cholesterol as a marker of T cell activation.

[0093] Uptake of cholesterol in T cells isolated from EO771 or AT3 breast tumors and restimulated ex vivo in the presence of fluorescent 3-NBD-cholesterol was measured. Due to the location and orientation of the NBD tag, this fluorescent cholesterol models cholesterol orientation in membranes better than previous fluorescently tagged cholesterol analogs. Solid tumors rarely contain naive T cells thus ex-vivo tumor infiltrating T cells were stimulated with low level CD3E for a brief period for re-stimulation. After re-stimulation, uptake of 3-NBD-Cholesterol was assessed in combination with activation marker CD69 on T cells using spectral cytometry (Figure 2A & 2D). Cholesterol uptake correlated significantly with expression of the activation marker, CD69 (Figure 2B & 2C). Additionally, cholesterol uptake increased to a markedly greater extent in CD8+ and CD4+ T cells from EO771 tumors when compared to T cells from AT3 (non-responsive) tumors (Figure 2E & 2F).

[0094] To assess uptake of cholesterol in T cells in vivo, tumor bearing mice were injected with a fluorescent BODIPY labeled cholesterol. BODIPY labeled cholesterol effectively labels intracellular cholesterol; remains stable in vivo; and exhibits greater fluorescence than the 3- NBD label. Twenty-four hours after injecting BODIPY-cholesterol, intra-tumoral T cells were collected for flow cytometry. T cells from responsive EO771 tumors showed a higher uptake of labeled cholesterol when compared to AT3 tumors as seen in histograms (Figure 3A & 3B) and normalized BODIPY MFI in both CD8+ and CD4+ T cells (Figure 3C & 3D). Prior studies reported increased expression of PD-1 on T cells that take up more cholesterol. Indeed, PD-1 expression on T cells increased significantly on CD8+ T cells from EO771 tumors with higher cholesterol uptake (Figure 3E). Modulation of PD-1 did not occur on CD4+ T cells (Figure 3F). Together these experiments demonstrate increased uptake of cholesterol in T cells from tumors that respond to immunotherapy.Example 2Activated T cells increased cholesterol uptake.

[0095] Cholesterol is required for cell division processes, and its presence in the plasma membrane of T cells stabilizes TCR nanoclusters to enhance overall activation. Cholesterol uptake in T cells remains a controversial topic. One study found that hypercholesteremia in T cells reflected proliferation of T cells. In contrast, elevated cholesterol in T cells also reportedly reflects exhaustion. To further investigate to what extent levels of cholesterol change with activation, naive mouse T cells were treated with increasing amounts of anti- CD3E and proportional increases in anti-CD28 antibodies (3 times the anti-CD3c concentration). Using an in vitro assay to quantify total cholesterol, free cholesterol, and cholesterol esters, it was established that activation increased total cholesterol concentration in T cells with significant differences between Opg / mL and 1 pg / mL or 10 pg / mL of anti- CD3e (p = 0.0033 and p = 0.0027, respectively) (Figure 4A). The concentration of cholesterol esters did not change with increasing activation in T cells after 24 hours (Figure 4A). While this assay demonstrates that total cholesterol increased in T cells after 1 day of TCR activation, these data do not distinguish between synthesis and uptake of cholesterol.

[0096] To selectively measure uptake of cholesterol during T cell activation, [18F]NP-59 was added to the culture medium. Thus, any accumulated radiotracer in T cells represents uptake, rather than de novo synthesis, during activation. Activated T cells were incubated with [18F]NP-59 according to the diagram in Figure 4B. After washing cells to remove extracellular radiotracer, autoradiography with a phosphor imaging screen was used to measure uptake by T cells. After one hour of incubation, uptake of [18F]NP-59 increased progressively with higher concentrations of activating antibodies. Each T cell treatmentdisplayed significant difference between levels of anti-CD3 stimulation with unique EC50 values when tested against all data points in logistical nonlinear regression (Figure 4C).

[0097] To extend these findings to humans, activated human T cells from peripheral blood were treated with [18F]NP-59 as outlined in Figure 4B. It was found human T cells also take up [18F]NP-59 when activated, but higher activation of anti-CD3 was stymied when compared to mice (Figure 4D). To study further the impact of cholesterol trafficking in the human immune system data from the Immune Cell Atlas, originating from Martin, et. al. 2019 and hosted by singlecell.broadinstitute.org was analyzed. These data comprise an annotated set of immune cells from an uninflamed lamina propria, an excellent tissue site for studying varied immune responses without the presence of disease. T cell clusters previously annotated by gene expression (Figure 4E) were used from this data. Genes responsible for uptake and trafficking of cholesterol displayed increased relative expression in the ‘Cycling’ cluster compared to other clusters (Figure 4F). These included genes specifically annotated for cholesterol uptake and trafficking, including scavenger receptors like SCP2 and SCARB1 , endosome associated proteins such as STARD3 / NL, NPC1 / 2, and transcription factor SREBF2. Additional upregulated genes, COMMD1 , WASH1 , ANXA2, and LAMTOR1 , function in cholesterol uptake and trafficking as well as other cellular processes. Increased expression of genes related to cholesterol uptake and trafficking in activated human T cells supports translatability of these findings.Example 3Uptake of [18F]NP-59 in T cells predicts and monitors response to immunotherapy in mouse TNBC

[0098] To test uptake of [18F]NP-59 in T cells in vivo and use of this radiotracer to predict and monitor response to immunotherapy, mice with EO771 or AT3 breast tumors were established. After tumors reached ~ 1 cm diameter, mice were randomly assigned to treatment with anti-PD1 or vehicle. It was believed that anti-PD-1 treatment would further alter cholesterol uptake with reinvigoration of endogenous T cells. Four days later, mice were intravenously injected with 1 OOpCi of [18F]NP-59. Uptake of [18F]NP-59 was quantified in total dissociated splenocytes, and T cells recovered from dissociated tumors. Splenocytes from mice treated with vehicle only accumulated comparable amounts of [18F]NP-59 for both EO771 and AT3 groups (Figure 5A). Relative to vehicle only, anti-PD1 treatment modestly increased radiotracer uptake in splenocytes from both groups, although amounts did not differ significantly. Isolated T cells from the vehicle treated EO771 tumors accumulated more [18F]NP-59 than vehicle treated AT3 tumors, reproducing results with fluorescent cholesterolanalogs (Figure 5B). Furthermore, T cells recovered from the anti-PD1 treated EO771 tumors had significantly greater uptake of [18F]NP-59 than anti-PD1 treated AT3 tumors (Figure 5C). These experiments demonstrate that baseline uptake of18[F]NP-59 before ICI treatment corelates with response to therapy in these models; and change in uptake of this radioactive cholesterol analog reflects T cell response to ICI.

[0099] To translate these discoveries to human breast cancer, publicly available datasets were queried and analyzed single cell RNA sequencing data from human TNBC. Louvain clusters of TNBC patient cells, without additional characterization or bias, reported four major T cell states (Figure 6A). These states included resting / naive T cells, highly activated / cycling T cells, transitional effectors (toward exhaustion), and exhausted effectors. T cell states were annotated based on highly expressed genes in each Louvain cluster: 1) Cycling T cells are defined by the high expression of genes related to microtubule reorganization and mitosis including upregulation of STNM1 and MKI67; 2) Transitional state cytotoxic T cells based on high expression of GZMK and moderate levels of PDCD1 and HAVRC2; 3) Dysfunctional T cells classified by high expression of PDCD1 , HAVCR2, CXCL13, and expression of CSF1 ; and 4) Resting memory T cells defined by high expression of CXCR4 and high expression of ribosomal proteins[31-33]. Cell counts of clusters from each patient revealed that patients with large T effector populations (whether transitional or exhausted) also carried a smaller population of actively proliferating T cells (Figure 6B). T cell states of each cluster were determined and annotated using top gene expression of the Louvain clusters (figure 4B). Cycling T cells displayed the greatest expression of genes directly and indirectly involved in cholesterol uptake and trafficking, including SREBP1 , master transcriptional regulator of cholesterol trafficking and metabolism. When analyzed using Enrichr families of genes were found related to “Regulation Of Cholesterol Biosynthesis By SREBF” and “Activation Of Gene Expression By SREBF” are significantly expressed in the ‘cycling’ T cell cluster (Figure 7). These data show some patients with TNBC, develop tumors that produce a population of cycling T cells that are accompanied by an expanded population of effector T cells, with features of gene expression indicative of cholesterol uptake and trafficking. Together, these data suggest that increased uptake of cholesterol demarks activated, anti-tumor T cells in human TNBC.Prophetic Example 1

[0100] Correlation of a change in uptake of a cholesterol analog imaging probe to responsiveness to an ICI therapy is evaluated on a full tumor basis. Multiple cell types, including cancer cells, T cells, and macrophages, in a tumor potentially may take up extracellular cholesterol. Reductions in immuosuppressive macrophage and / or cancer cells may cause the overall whole tumor uptake of [18F]NP-59 to decrease as a marker ofsuccessful immunotherapy. Alternatively, increases in number and activation status of T cells could produce an increase in cholesterol during successful immunotherapy. To assess these alternatives, we will quantify whole tumor uptake of [18F]NP-59 in E0771 breast tumors treated with aniti-PD-1 immunotherapy as compared with control. These data will indicate to what extent changes in uptake of [18F]NP-59 either up or down denote successful therapy on a whole tumor basis as would be assessed by PET imaging.

[0101] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the disclosure may be apparent to those having ordinary skill in the art.

[0102] All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In the case of conflict, the present description, including definitions, will control.

[0103] Throughout the specification, where the compounds, compositions, methods, and / or processes are described as including components, steps, or materials, it is contemplated that the compounds, compositions, methods, and / or processes can also comprise, consist essentially of, or consist of any combination of the recited components or materials, unless described otherwise. Component concentrations can be expressed in terms of weight concentrations, unless specifically indicated otherwise. Combinations of components are contemplated to include homogeneous and / or heterogeneous mixtures, as would be understood by a person of ordinary skill in the art in view of the foregoing disclosure.References1. Bagegni NA, Davis AA, Clifton KK, Ademuyiwa FO. Targeted Treatment for High-Risk Early-Stage Triple-Negative Breast Cancer: Spotlight on Pembrolizumab. Breast Cancer (Dove Med Press). 2022;14:113-23.2. Oshi M, Newman S, Tokumaru Y, Yan L, Matsuyama R, Endo I, et al. 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Claims

What is claimed is:1 . A method of predicting responsiveness of a disease to treatment with an immune checkpoint inhibitor, comprising: contacting a sample comprising T-cells with a cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the T- cells; and imaging the contacted sample for detection of an uptake of the cholesterol analog imaging probe by the T-cells, wherein a detectable uptake of the cholesterol analog imaging probe is predictive of the cancer being responsive to treatment with the immunotherapy, wherein the cholesterol analog imaging probe has a label selected from a halogen or a radiohalogen.

2. A method of evaluating responsiveness of a disease to treatment with an immune checkpoint inhibitor, comprising: contacting a pre-treatment sample with a cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by cells present in the sample, wherein the pre-treatment sample is obtained from a subject prior to administration of treatment; imaging the contacted pre-treatment sample for detection of an uptake of the cholesterol analog imaging probe by the cells in the pre-treatment sample, contacting a post-treatment sample comprising cells with the cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the post-treatment sample is obtained from the subject after treatment for the treatment duration; and imaging the contacted post-treatment sample for detection of an uptake of the cholesterol analog imaging probe by the cells, wherein a change in the detected uptake of the cholesterol analog imaging probe in the post-treatment sample relative to the pre-treatment sample is predictive of the cancer being responsive to treatment with the immunotherapy, wherein the cholesterol analog imaging probe has a label selected from a halogen or a radiohalogen.

3. The method of claim 2, wherein the treatment duration is at least two weeks.

4. The method of claim 1 or 2, wherein the sample is a tumor sample, a blood sample, or a biopsy sample.

5. The method of claim 1 or 2, wherein imaging the contacted sample is performed using one or more of positron emission tomography (PET), positron emission tomography / computed tomography (PET / CT), positron emission tomography / magnetic resonance imaging (PET / MRI), single-photon emission computerized tomography (SPECT), and single-photon emission computerized tomography / computed tomography (SPECT / CT).

6. The method of any one of claims 1 to 4, wherein the disease is a cancer or an autoimmune disease or disorder.

7. The method of claim 5, wherein the disease is cancer selected from lung cancer, cervical cancer, colon cancer, bladder cancer, breast cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, skin cancer, stomach cancer, or rectal cancer.

8. The method of claim 6, wherein the cancer is triple negative breast cancer.

9. The method of any one of the preceding claims, wherein the label is I, F, Cl, or Br, or a radioisotope thereof.

10. The method of any one of the preceding claims, wherein the immune check point inhibitor an anti-PD1 antibody or an anti-PDL1 antibody or an anti-CTLA-4 antibody.

11. A method of predicting responsiveness of a subject suffering from a cancer or autoimmune disease to treatment with an immune checkpoint inhibitor, comprising: administering to the patient a cholesterol analog imaging probe; imaging one or more cancerous regions of the patient or imaging one or more regions of the patient affected by autoimmunity for an uptake of the cholesterol analog imaging probe in the one or more cancerous regions, wherein a detectable uptake of the cholesterol analog imaging probe is predictive of the cancer being responsive to treatment with the immunotherapy, and wherein the cholesterol analog imaging probe has a label is a halogen or a radiohalogen.

12. The method of claim 10 or 1 1 , wherein the one or more cancerous regions comprises a tumor.

13. The method of claim 10 or 11 , further comprising performing a biopsy of the tumor and performing an ex vivo evaluation of the cholesterol analog imaging probe uptake on the biopsy.

14. The method of any one of claims 10 to 13, wherein the cancer is lung cancer, cervical cancer, colon cancer, bladder cancer, breast cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, skin cancer, stomach cancer, or rectal cancer15. The method of claim 14, wherein the cancer is triple negative breast cancer.

16. The method of claim 10 to 15, wherein the immune check point inhibitor is an anti-PD1 antibody or an anit-PDL1 antibody or an anti-CTLA-4 antibody.

17. A method of evaluating responsiveness to treatment with an immune checkpoint inhibitor in a subject in need thereof, comprising: contacting a first sample comprising cells with a cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the first sample is obtained prior to administration of the immunotherapy treatment; imaging the contacted first sample for detection of an uptake of the cholesterol analog imaging probe by the cells; contacting a second sample comprising cells with a cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the second sample is obtained after administration of an immunotherapy; imaging the contacted second sample for detection of an uptake of the cholesterol analog imaging probe by the cells; and comparing the uptake of the cholesterol analog imaging probe in the first sample to the uptake of the cholesterol analog imaging probe in the second sample, wherein a change in uptake in the second sample as compared to the first sample is indicative of responsiveness to treatment with the immunotherapy, and wherein the cholesterol analog imaging probe has a label is a halogen or a radiohalogen.

18. The method of claim 17, wherein the cells comprise one or more of T -cells, macrophages, and tumor cells.

19. The method of claim 17 or 18, wherein the sample is a blood sample, a biopsy sample, or a tumor sample.

20. The method of claim 17 or 19, wherein the subject suffers from cancer or an autoimmune disorder or disease.21 . The method of claim 20, wherein the subject suffers from cancer selected from lung cancer, cervical cancer, colon cancer, bladder cancer, breast cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, skin cancer, stomach cancer, or rectal cancer22. The method of claim 21 , wherein the cancer is triple negative breast cancer.

23. The method of any one of claims 17 to 23, wherein the immune check point inhibitor is an anti-PD1 antibody or anti-PDL1 Antibody or an anti-CTLA-4 antibody.

24. A method of evaluating responsiveness to treatment with an immune checkpoint inhibitor in a subject in need thereof, comprising: administering a cholesterol analog imaging probe to the subject prior to administration of the immune checkpoint inhibitor; imaging the subject to evaluate an uptake of the cholesterol analog imaging probe in one or more cancerous regions or one or more regions affected by autoimmunity before treatment; administering the cholesterol analog imaging probe after administration of the immune checkpoint inhibitor; imaging the subject to evaluation an uptake of the cholesterol analog imaging probe in the one or more cancerous regions or the one or more regions affected by autoimmunity after treatment; comparing the uptake of the cholesterol analog imaging probe before treatment to the uptake of the cholesterol analog imaging probe after treatment, wherein a change in uptake is indicative of the responsiveness of the subject to treatment with the immune checkpoint inhibitor, wherein the cholesterol analog imaging probe has a label or a halogen radiohalogen.

25. The method of claim 24, wherein the one or more cancerous regions comprises a tumor.

26. The method of claim 25, further comprising performing a biopsy of the tumor and performing an ex vivo evaluation of the cholesterol analog imaging probe uptake on the biopsy.

27. The method of any one of claim 24 to 26, wherein the cancer is lung cancer, cervical cancer, colon cancer, bladder cancer, breast cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, skin cancer, stomach cancer, or rectal cancer28. The method of claim 27, wherein the cancer is triple negative breast cancer.

29. The method of any one of claims 24 to 28, wherein the immune check point inhibitor is an anti-PD1 antibody or an anti-PDL1 antibody an anti-CTLA-4 antibody.

30. The method of any one of claims 17 to 29, wherein the change in uptake of the cholesterol analog imaging probe is a change in a standardized uptake value of at least 0.5.31 . The method of any one of the preceding claims, wherein the cholesterol analog imaging probe is a compound of formula I:R1is OH or OP;R2, when present, is OH or X;R3is H, OH, X, CH2-X, or CH2-LG;R4, when present, is C1-6 alkyl, C1-6 alkylene-X, or C1-6 alkylene-LG;X is a halogen or radiohalogen;P is an alcohol protecting group;LG is a leaving group; each of bond A and bond B is a single or a double bond and only one of bond A and bond B can be a double bond, with the proviso that at least one X as the label.

32. The method of claim 31 , wherein if one of R2and R3is F and the other OH, then the F is18FThe method of any one of the preceding claims, wherein the cholesterol analog imaging probe has a structure selected from the group consisting of:

33. The method of any one of the preceding claims, wherein the cholesterol analog34. The method of any one of the preceding claims, wherein the cholesterol analog35. A cholesterol analog imaging probe for use in predicting responsiveness of a disease to treatment with an immune checkpoint inhibitor, the use characterized in that predicting responsiveness comprises: contacting a sample comprising T-cells with the cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the T- cells; and imaging the contacted sample for detection of an uptake of the cholesterol analog imaging probe by the T-cells, wherein a detectable uptake of the cholesterol analog imaging probe is predictive of the cancer being responsive to treatment with the immunotherapy, wherein the cholesterol analog imaging probe has a label selected from a halogen or a radiohalogen.

36. A cholesterol analog imaging probe for use in evaluating responsiveness of a disease to treatment with an immune checkpoint inhibitor, characterized in that the evaluating comprising: contacting a pre-treatment sample with the cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by cells present in the sample, wherein the pre-treatment sample is obtained from a subject prior to administration of treatment;imaging the contacted pre-treatment sample for detection of an uptake of the cholesterol analog imaging probe by the cells in the pre-treatment sample; contacting a post-treatment sample comprising cells with the cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the post-treatment sample is obtained from the subject after treatment for the treatment duration; and imaging the contacted post-treatment sample for detection of an uptake of the cholesterol analog imaging probe by the cells, wherein a change in the detected uptake of the cholesterol analog imaging probe in the post-treatment sample relative to the pre-treatment sample is predictive of the cancer being responsive to treatment with the immunotherapy, wherein the cholesterol analog imaging probe has a label selected from a halogen or a radiohalogen.

37. A cholesterol analog imaging probe for use in predicting responsiveness of a subject suffering from a cancer or autoimmune disease to treatment with an immune checkpoint inhibitor, characterized in that the predicting comprising: administering to the patient the cholesterol analog imaging probe; imaging one or more cancerous regions of the patient or imaging one or more regions of the patient affected by autoimmunity for an uptake of the cholesterol analog imaging probe in the one or more cancerous regions, wherein a detectable uptake of the cholesterol analog imaging probe is predictive of the cancer being responsive to treatment with the immunotherapy, and wherein the cholesterol analog imaging probe has a label is a halogen or a radiohalogen.

38. A cholesterol analog imaging probe for use in evaluating responsiveness to treatment with an immune checkpoint inhibitor in a subject in need thereof, characterize in that the evaluating comprising: contacting a first sample comprising cells with the cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the first sample is obtained prior to administration of the immunotherapy treatment; imaging the contacted first sample for detection of an uptake of the cholesterol analog imaging probe by the cells;contacting a second sample comprising cells with a cholesterol analog imaging probe under conditions sufficient to allow uptake of the cholesterol analog imaging probe by the cells, wherein the second sample is obtained after administration of an immunotherapy; imaging the contacted second sample for detection of an uptake of the cholesterol analog imaging probe by the cells; and comparing the uptake of the cholesterol analog imaging probe in the first sample to the uptake of the cholesterol analog imaging probe in the second sample, wherein a change in uptake in the second sample as compared to the first sample is indicative of responsiveness to treatment with the immunotherapy, and wherein the cholesterol analog imaging probe has a label is a halogen or a radiohalogen.

39. A cholesterol analog imaging probe for use in evaluating responsiveness to treatment with an immune checkpoint inhibitor in a subject in need thereof, characterized in that the evaluating comprising: administering the cholesterol analog imaging probe to the subject prior to administration of the immune checkpoint inhibitor; imaging the subject to evaluate an uptake of the cholesterol analog imaging probe in one or more cancerous regions or one or more regions affected by autoimmunity before treatment; administering the cholesterol analog imaging probe after administration of the immune checkpoint inhibitor; imaging the subject to evaluation an uptake of the cholesterol analog imaging probe in the one or more cancerous regions or the one or more regions affected by autoimmunity after treatment; comparing the uptake of the cholesterol analog imaging probe before treatment to the uptake of the cholesterol analog imaging probe after treatment, wherein a change in uptake is indicative of the responsiveness of the subject to treatment with the immune checkpoint inhibitor,

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