Methods of detecting biomarkers by fluorescence imaging

Abstract

Methods detect or quantify biomarkers such as Programmed Death-Ligand 1 (PD-L1) or immune checkpoint biomarkers in tissue of a sample or a subject. Methods predict or monitor response by a subject to cancer immunotherapy by detecting or quantifying immune checkpoint biomarkers. Methods detect or quantify a panel of biomarkers in tissue by contacting the tissue with a set of targeted and untargeted fluorescent agents and performing spectral unmixing of the fluorescence emission data.

Description

METHODS OF DETECTING BIOMARKERS BY FLUORESCENCE IMAGINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 764,563, filed on February 27, 2025, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to methods of detecting biomarkers by fluorescence imaging. The disclosure also relates to methods of detecting a Programmed Death-Ligand 1 axis protein in tissue, and to methods of predicting or monitoring response by a subject to cancer immunotherapy.BACKGROUND

[0003] Immune checkpoint inhibitors (ICIs) have significantly improved overall survival in multiple difficult-to-treat cancers. ICIs target the interaction between immune-inhibitory receptors, including programmed cell death protein 1 (PD1) on immune cells and its primary ligand, Programmed Death-Ligand (PDL1 or PD-L1), broadly found on tumor, normal, and immune cells. Additionally, PDL1 can bind to cluster of differentiation 80 (CD80), further modulating immune responses. Several PDL1 assays have been approved as a companion or complementary diagnostic for ICIs depending on the cancer subtype. However, tumor PDL1 expression measured by standard approaches alone cannot reliably stratify patients into responders or non-responders. A significant limitation of many current assays for biomarkers lies in their reliance on tissue biopsies, which provide only a static snapshot of tumor PDL1 status and its immune state. Patients are generally administered given anti-PDl ICIs based on either tumor PDL1 expression or degree of tumor mutational burden. However, relying on tumor PDL1 expression or mutational burden alone does not fully capture the functional status or dynamic nature of these immune checkpoints.

[0004] Immunohistochemical (IHC) staining of primary tumors offers static snapshots of PDL1 presence but fails to dynamically assess receptor availability in real-time or across different tissue sites. Several FDA-approved PDL1 assays serve as companion diagnostics to ICIs and are based on a scoring system that sums PDL1 -positive cells on tissue biopsies.However, in a systemic review investigating the primary studies that supported 45 FDA drug approvals across 15 tumor types, PDL1 expression was found to be predictive in only 28.9% of cases. Davis et al., J Jmm another Cancer 7, 278 (2019). Current in vivo imaging techniques cannot dynamically measure the precise, real-time availability of immune checkpoints and their binding partners. Other analytical techniques, such as flow cytometry, can provide information on the relative levels of cell-specific protein expression but are limited to ex vivo analysis and lack spatial information on cell-to-cell interactions within tissues.

[0005] Paired agent imaging (PAI) can overcome high background noise observed in singletargeted fluorescence imaging by using a targeted agent that binds a protein of interest and an untargeted agent to correct non-specific (unbound) signals. See, e.g., Tichauer et al., In Vivo Quantification of Tumor Receptor Binding Potential with Dual-Reporter Molecular Imaging. Mol Imaging Biol 14, 584-592 (2012); Tichauer et al., Improved tumor contrast achieved by single time point dual-reporter fluorescence imaging. J. Biomed. Opt. 17, 066001 (2012);Samkoe et al., Quantitative In Vivo Immunohistochemistry of Epidermal Growth Factor Receptor Using a Receptor Concentration Imaging Approach. Cancer Research 74, 7465-7474 (2014); Samkoe et al., Paired-agent imaging for detection of head and neck cancers, Proc SPIE Int Soc Opt Eng. 2019 Feb;10853: 108530P. doi: 10.1117 / 12.2510897. The resulting PAI signal, called the binding potential (BP), is proportional to the targeted receptor density multiplied by the binding affinity of the targeted agent to the protein of interest.

[0006] Multi-channel fluorescence imaging systems use a set of channels optimized for single excitation and emission wavelengths. For in vivo applications, they offer high sensitivity to fluorescence signals but can restrict the use of multiple fluorescent markers with spectral overlap. Multi-channel fluorescence imaging systems also inadvertently capture tissue autofluorescence from samples, interfering with signal detection and accuracy of quantitative data on the fluorescence target. Multi-spectral imaging systems can address these challenges by simultaneously acquiring data across multiple spectral bands to differentiate overlapping fluorescence signals. Multi-spectral imaging provides a more time- and cost-effective approach for spectrally characterizing and removing autofluorescence.

[0007] There is an unmet need for methods that can accurately detect or quantify immune checkpoint biomarkers, such as PD1, PDL1, and CD80, including in in vivo tissue. There is alsoan unmet need for methods that can accurately detect or quantify a panel of biomarkers in tissue using multiple fluorescent dyes.SUMMARY

[0008] As one aspect of the present disclosure, methods are provided for detecting or quantifying biomarkers such as Programmed Death-Ligand 1 (PD-L1 or PDL1) in tissue of a sample or a subject. The tissue is contacted with a targeted fluorescent agent that binds to a biomarker. The tissue is illuminated one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent. Targeted fluorescence emission data is acquired for one or more emission wavelengths associated with the first targeted fluorescent agent. The biomarker in the tissue is detected or quantified based on the targeted fluorescence emission data. In some embodiments, the methods employ Paired Agent Imaging (PAI).

[0009] As another aspect of the present disclosure, methods are provided for predicting or monitoring response by a subject to cancer immunotherapy. The methods comprise detecting or quantifying Programmed Death-Ligand (PD-L1 or PDL1) in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to PD-L1. A PD-L1 score is determined for the tissue and is used for predicting or monitoring response by a subject to cancer immunotherapy. In some embodiments, the methods comprise detecting or quantifying available Programmed Death-Ligand (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll).

[0010] As yet another aspect of the present disclosure, methods are provided for detecting or quantifying a panel of biomarkers in tissue of a sample or subject. For example, the panel of biomarkers can comprise the PD-L1 axis proteins (PD-L1, PD-1 and CD80). The methods comprise contacting the tissue with a set of targeted fluorescent agents that bind to the biomarkers and a non-targeted fluorescent agent, wherein each of the targeted fluorescent agents and the non-target fluorescent agent has a different emission spectrum. The tissue is illuminated one or more times with light of stimulus wavelength(s) absorbed by each of the targeted fluorescent agents and the non-targeted fluorescent agent. Targeted fluorescence emission data is acquired for one or more emissions wavelengths associated with each of the targeted fluorescent agents. Non-targeted fluorescence emission data is acquired for one or more emissions wavelengths associated with the non-targeted fluorescent agent. The methods also comprise performing spectral unmixing of the targeted fluorescence emission data and non-targeted fluorescence emission data. The targeted fluorescence emission data is corrected based on the non-targeted fluorescence data. Each of the biomarkers of the panel in the tissue is detected or quantified based on the spectrally-unmixed corrected targeted fluorescence emission data.

[0011] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment s) and together with the description, serve to explain the principles described herein.

[0013] FIG. 1 illustrates measurement of dissociation constants of imaging agents to the PDL1 axis proteins in a syngeneic murine lymphoma cell line, (a) Saturation curve of anti-PDLl antibody, (b) anti-PDl antibody, and (c) anti-CD80 antibody binding on E.G7-OVA cells analyzed by flow cytometry. Panels (a - c) show median fluorescence intensity values across varying antibody concentrations from three independent experimental trials, (d) Dissociation constants for anti-PDLl, anti-PDl, and anti-CD80 antibodies calculated using one-site specific binding model from saturation curves. Error bars represent the standard deviation, ns = not significant by unpaired, two-tailed t-test.

[0014] FIG. 2 illustrates quantification of PDL1 axis proteins per E.G7-OVA cell, (a) Representative schematic of QFC, including histograms of CD80 expression on E.G7-OVA cells using AF488-labelled anti-CD80 antibody and calibration of AF488 MESF beads for quantification, (b) PDL1, PD1, and CD80 receptors in E.G7-OVA cells measured by QFC. Error bars represent the standard deviation. ***p < 0.001 by one-way ANOVA.

[0015] FIG. 3 summarizes selection of fluorophores for multi-spectral paired agent imaging, (a) Excitation (dashed lines) and emission (solid lines) spectra of IRD680, AF700, AF750, and IRD800. (b) Mixed spectra of all dyes in a liquid phantom with 1% intralipid and 1% BSA (Left). Unmixed spectra of each fluorophore and Intralipid / BSA (Right), (c) Fluorescent signal intensity of IRD680, AF700, and AF750 either when alone or in four-dye mixtures after spectral unmixing. ****p < 0.0001, ns = not significant by unpaired two-tailed t-test.

[0016] FIG. 4 illustrates the determination of suitable incubation time for targeted and untargeted fluorescent agents for a tumor-mimicking phantom model using two-dimensional mean squared displacement, (a) Diffusion coefficients of imaging agents (PDL1-AF750, PD1-IRD680, CD80-AF700, and IgG-IRD800) determined from agar experiments, (b) Table summarizing the diffusion coefficient and experimental time required for each imaging agent to evenly distribute through a 0.106 cm deep E.G7-OVA tumor-mimicking phantom. Panels (a-b) show the mean and standard deviation values from three independent experimental trials, (c) E.G7-OVA cells were first embedded in 0.3% agar (a representative brightfield image is shown). ***p < 0.001, **P < 0.01, *P <0.05, ns = not significant by unpaired, two-tailed t-test.

[0017] FIG. 5 illustrates that multi-spectral paired agent imaging of receptors (mPAIR) quantifies the receptor concentrations of PDL1 axis proteins in a tumor-mimicking phantom model, (a) Representative workflow of concurrent mPAIR and QFC experiments. E.G7-OVA cells were collected and split into two groups: the first was used in E.G7-OVA tumormimicking phantoms, and the second was used to measure PDL1, PD1, and CD80 receptors via QFC. (b) Representative plot of spectrally unmixed fluorescent signals from all imaging agents in a E.G7-OVA tumor-mimicking phantom, (c) Mixed spectral image cube and spectrally unmixed images of an E.G7-OVA tumor-mimicking phantom at the 120-minute time point post-rinse, (d) STP BP maps at the 120-minute time point for each targeted receptor, (e) Linear correlation of the number of PDL1, PD1, and CD80 receptors in E.G7-OVA cells determined by mPAIR (in tumor phantoms) and QFC run in parallel (n=3 independent trials). Pearson correlation (r), bias correction factor (Cb), Lin’s CCC (pc), and RMSE was calculated between the two methods. The line of identity is represented on the scatter plot with a black dashed line. Data and images presented in (b-d) are from a single trial.

[0018] FIG. 6 shows UV-vis spectroscopy data of fluorescently tagged antibodies in panels (a.g). UV-vis spectroscopy of (a) PDL1-AF488, (b) PD1-AF488, (c) CD80-AF488, (d) PDL1-AF750, (e) PD1-IRD680, (f) CD80-AF700, and (g) IgG-IRD800. Panel (h) is a table listing dye to protein ratios of labelled antibodies determined from UV-vis spectroscopy data.

[0019] FIG. 7 illustrates spectral unmixing data in log scale for single and combination liquid phantom experiments. Fluorophore signal intensity in log scale of IRD680, AF700, AF750, IRD800 when alone or in four-dye mixtures using reference spectral library (data from FIG.3(b)).

[0020] FTG. 8 shows representative acquisitions for PD1-IRD680 for the determination of diffusion coefficient using two-dimensional mean squared displacement. Fluorescent images (left) were rotated at (a) 0°, (b) 45°, (c) 90°, and (d) 135°. Intensity profiles derived from column-summed pixel values over time (center), and corresponding radii estimated from the full width at half maximum of each profile over time (right) are shown for each rotation. Slope of the linear relationship between distance travelled and time was used to determine the diffusion coefficient.

[0021] FIG. 9 illustrates representative rinsing paired-agent kinetic model (RPAM) curves for PDL1, PD1, and CD80. Time-dependent fluorescent signals of targeted imaging agents (PDL1-AF750, PD1-IRD680, CD80-AF700) and untargeted control (IgG-IRD800) are shown. RPAM curves are shown for (a) PDL1-AF750 imaging agent, (b) PD1-IRD680 imaging agent, and (c) CD80-AF700 imaging agent.

[0022] FIG. 10 is a Bland-Altman plot comparing mPAIR and QFC. The Bland-Altman plot compares log PDL1, PD1, and CD80 receptors per population determined by mPAIR and QFC. Bias is represented with a blue solid line, the upper 95% limit of agreement is represented with a black dotted line, and the lower 95% limit of agreement is represented with an orange dotted line.

[0023] FIG. 11 shows a representative E.G7OVA mouse used for mPAIR imaging, (a) Image of E.G7OVA tumor (T) in the flank region and adjacent leg muscle (M) exposed for mPAIR imaging, (b) Raw representative fluorescence spectra of E.G7OVA [1] LP tumor and muscle. Tumor and muscle autofluorescence spectra are the average from three E.G7OVA tumorbearing mice that were not administered with the mPAIR imaging cocktail.

[0024] FIG. 12 shows representative spectral unmixing of E.G7OVA tumor and muscle Regions of Interest (ROIs). (a) ROI selected from the tumor region, (b) Mixed fluorescence signals from the tumor ROI. (c) Spectrally unmixed signals of individual mPAIR imaging agents and tissue autofluorescence, (d) Unmixed integrated signals from the AUCs of spectra shown in (c). (e) ROI selected from the adjacent leg muscle region, (f) Mixed fluorescence signals from the muscle ROI. (g) Spectrally unmixed signal of individual mPAIR imaging agents and tissue autofluorescence, (h) Unmixed integrated signals from the AUCs of spectra shown in (g).Panels b - d represent data extracted from the same tumor ROI in panel (a). Panels f-h represent data extracted from the same tumor ROI in panel (e).

[0025] FTG. 13 shows (a) mixed spectral image cube of E.G7OVA tumor and muscle regions. (b) spectrally unmixed image cubes of an E.G7OVA tumor, (c) STP BP maps for each targeted receptor using the muscle region for normalization. Data and images presented in (a-c) are representative images from a single mouse.

[0026] FIG. 14 shows outer versus inner binding potentials (BPs) from E.G7OVA tumors, (a) Representative outer and inner ROIs for tumor. Pearson correlation (r) of the BPs from the outer region vs. inner region for (b) PDL1, (c) PD1, and (d) CD80.

[0027] FIG. 15 shows Spearman correlation (p) of the (a-c) number of PDL1, PD1, and CD80 receptors and (c) log-transformed values in E.G7OVA tumors determined by mPAIR and QFC.

[0028] FIG. 16 shows (a) immunofluorescence (IF) images of tumor tissue sections taken at 20x magnification, with two representative fields shown for both inner and outer tumor regions, (b) Normalized FMI signal intensities for PDL1, PD1, and CD80 in inner and outer tumor regions. (c) Median receptors per population from the corresponding mice determined from QFC. Panels a-c represent images and data from one mouse.

[0029] FIG. 17 shows residual imaging agent signal in E.G7OVA tumors from mPAIR-administered mice, (a) Spectrally unmixed signals for mPAIR dyes detected in tumor cells using the Aurora flow cytometry software. A single stained spleen sample labelled with only Live / Dead Blue viability dye (L / D blue) served as the fluorescence-minus-one (FMO) control. Comparisons between (b) AF750 signal intensity and PDL1 receptors, (c) IRD680 signal intensity and PD1 receptors, and (d) AF700 signal intensity and CD80 receptors. Data in panels a-d are from the third cohort of mPAIR mice.

[0030] FIG. 18 compares the receptors per population quantified in the pan immune versus mPAIR cohorts for (a) PDL1, (b) PD1, and (c) CD80. Error bars represent the standard deviation, ns = not significant, ****p < 0.0001 ***p < 0.001 by unpaired t-test.

[0031] FIG. 19 shows violin plots showing the distribution of (a) binding potentials from mPAIR-administered mice and (b) log-transformed receptors per population from pan immune mice across individual tumors.

[0032] FIG. 20 shows (a) representative tumor ROI from E.G7OVA [2] LP mouse. Pearson correlation (r) of (b) PD1 colocalization with PDL1 BPs, (b) CD80 colocalization with PDL1 BPs, and (d) PD1 colocalization with CD80 BPs. Data is shown of one representative mouse.

[0033] FTG. 21 shows (a) table listing Pearson correlations of BP colocalization of receptors in E.G7OVA mice, (b) violin plot of Pearson correlations listed in (a).

[0034] FIGs. 22A-C show representative confocal images of soluble PD1 binding (22A), anti-PDL1 binding (22B), and colocalization of soluble PD1 and anti-PDLl antibody (22C) on melanoma tumors.

[0035] FIGs. 23A-F demonstrate that PAFI quantified unbound, functional PDL1 receptors in lymphoma and macrophage-like tumor cells using soluble PD1.

[0036] The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale. Wherever practical, like reference numerals refer to like features.DETAILED DESCRIPTION

[0037] As one aspect of the present disclosure, methods are provided for detecting or quantifying biomarkers in tissue of a sample or a subject. The tissue is contacted with a targeted fluorescent agent that binds to a biomarker. The tissue is illuminated one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent. Targeted fluorescence emission data is acquired for one or more emission wavelengths associated with the first targeted fluorescent agent. The biomarker in the tissue is detected or quantified based on the targeted fluorescence emission data.

[0038] In some embodiments, the methods employ Paired Agent Imaging (PAI). To that end, the methods can further comprise contacting the tissue with a non-targeted fluorescent agent in addition to the targeted fluorescent agent. As used here, “non-targeted” and “untargeted” are used interchangeably unless the context indicates otherwise. The tissue is illuminated one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent. Non-targeted fluorescence emission data is acquired for one or more emissions wavelengths associated with the non-targeted fluorescent agent. The targeted fluorescence emission data is corrected using the non-targeted fluorescence data, and biomarker in the tissue is detected or quantified based on the corrected targeted fluorescence emission data.

[0039] As yet another aspect of the present disclosure, methods are provided for detecting or quantifying a panel of biomarkers in tissue of a sample or subject. The methods comprise contacting the tissue with a set of targeted fluorescent agents that bind to the biomarkers and anon-targeted fluorescent agent, wherein each of the targeted fluorescent agents and the nontarget fluorescent agent has a different emission spectrum. The tissue is illuminated one or more times with light of stimulus wavelength(s) absorbed by each of the targeted fluorescent agents and the non-targeted fluorescent agent. Targeted fluorescence emission data is acquired for one or more emissions wavelengths associated with each of the targeted fluorescent agents. Non-targeted fluorescence emission data is acquired for one or more emissions wavelengths associated with the non-targeted fluorescent agent. The methods also comprise performing spectral unmixing of the targeted fluorescence emission data and non-targeted fluorescence emission data. The targeted fluorescence emission data is corrected based on the non-targeted fluorescence data. Each of the biomarkers of the panel in the tissue is detected or quantified based on the spectrally-unmixed corrected targeted fluorescence emission data.

[0040] The present methods can be used for detecting or quantifying one or more biomarkers present in a tissue. Examples of biomarkers include Programmed Death-Ligand 1 (PD-L1) axis proteins, such as PDL1, PD1, and CD80. In some embodiments, the present methods comprise detecting or quantifying available Programmed Death-Ligand 1 (PD-L1) and / or Functional Programmed Death-Ligand 1 (f-PD-Ll). Available Programmed Death-Ligand 1 (a-PD-Ll) includes PD-L1 available for binding within the tumor microenvironment, such as available for binding to immune checkpoint inhibitors (ICIs). Functional Programmed Death-Ligand 1 (f-PD-Ll) includes PD-L1 that is able to transmit an intracellular or other biological signal. In some contexts or embodiments, PD-L1 may be unavailable or non-functional due to PD-1 or CD80 binding, lack of glycosylation, and / or other causes. In some contexts or embodiments of the present methods, a-PD-Ll and f-PD-Ll may be the same in kind or amount, while in other contexts or embodiments, a-PD-Ll and f-PD-Ll may be different quantities and / or refer to different sets of PD-L1 receptors. In such embodiments, the tissue can be contacted with a targeted fluorescent agent that binds to available Programmed Death-Ligand 1 (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll) and does not specifically bind to PD-L1 complexed with PD-1 or CD80.

[0041] Another aspect of the present disclosure provides methods for predicting or monitoring response by a subject to cancer immunotherapy. The methods comprise detecting or quantifying Programmed Death-Ligand (PD-L1) in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to PD-L1. A PD-L1 score is determined for thetissue and is used for predicting or monitoring response by a subject to cancer immunotherapy. In some embodiments, the methods comprise detecting or quantifying available Programmed Death-Ligand (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll).

[0042] The present disclosure provides methods that employ mPAIR as a novel in vivo multi-spectral imaging technique that quantifies the availability of the PDL1 axis proteins or other panels of biomarkers. By using a panel of NIR fluorophores to image PDL1, PD1, and CD80 simultaneously, mPAIR addresses the limitations of static biopsy-based and single protein biomarkers to evaluate ICI eligibility. The present disclosure demonstrates mPAIR’s ability to assess the spatial distribution of receptor concentrations with minimal spectral overlap.

[0043] It is also contemplated that the present mPAIR methods can be used to detect, quantify, and / or test the availability of other biomarkers (such as immune checkpoint receptors or ligands) to guide cancer immunotherapy decisions. Other immune checkpoints include CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4), LAG-3 (Lymphocyte-activation gene 3), TIM-3 (T cell immunoglobulin and mucin-domain containing-3), TIGIT (T cell immunoreceptor with Ig and ITEM domains), BTLA (B and T lymphocyte attenuator), VISTA (V-domain Ig suppressor of T cell activation), CD47, IDO1 (Indoleamine 2,3-dioxygenase 1), B7-H3 (B7 Homolog 3) or B7-H3 and B7-H4 (B7 Homolog 4). To detect, quantify, and / or test the availability, targeted fluorescent agents that bind to such immune checkpoints can be used in place of the exemplary targeted fluorescent agents disclosed herein.

[0044] ICIs are among the most effective classes of cancer immunotherapies and have improved overall survival in multiple advanced- stage cancers. While durable, long-term responses have been clinically reported in a subset of patients, many patients receiving ICIs fail to respond to treatment irrespective of their cancer histology. To address the limitations of current assays for biomarkers used in connection with cancer immunotherapy, the present disclosure describes in vivo imaging methods that quantify PDL1 and its cognate receptors, PD1 and CD80, and / or quantify PD-L1 receptors that are available for binding. mPAIR builds on previous advancements in PAI, quantitatively measures in vivo expression of a single protein. The present disclosure enables mPAIR to simultaneously measure the concentrations of multiple receptors in a tissue such as PDL1, PD1, and CD80 receptors. While PAI is conventionally used to measure one protein of interest, the present approach extends PAI into amulti-spectral PAI of receptors (mPATR) method to simultaneously quantify multiple proteins in vivo.

[0045] In some embodiments, the targeted fluorescent agent comprises a binding agent and a fluorophore. The binding agent is selected for its specific binding to a biomarker. For example, the binding agent can be selected from the group consisting of soluble proteins (such as soluble PD-1), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof. In some embodiments, the binding agents have similar Kdvalues ranging from 0.01 nM to 10 nM, alternatively from 0.1 nM to 1 nM, alternatively from 0.2 nM to 0.5 nM, alternatively from 0.30 nM to 0.38 nM. In some embodiments, the binding agents reach saturation at 10 nM, as shown in FIG. 1, indicating comparable binding characteristics.

[0046] In some embodiments, the non-targeted fluorescent agent comprises a non-specific agent and a fluorophore; in other embodiments, the non-targeted fluorescent agent comprises a fluorophore without a non-specific agent (for example, a non-targeted fluorescent agent can consist of a fluorophore only). The non-specific agent lacks specific binding to biomarkers in the tissue, but otherwise has comparable properties to the binding agent. In some embodiments, the binding agent and the non-specific agent have similar pharmacokinetic and physical properties (e.g., molecular weights, isoelectric points, clearance rate, stability), such as molecular weights or other properties within 10% of each other, alternatively within 5%, 2%, 1% or 0.1%. To that end, the non-specific agent can also be an antibody, antibody fragment, single chain antibody, single domain antibody, antibody mimetic, or mixture thereof, but its domains do not specifically bind receptors present in the tissue, or do not bind to a significant or appreciable degree. In some embodiments, the binding agent and non-specific agent comprise antibodies or fragments of the same clonal isotype. In some embodiments, the binding agent and non-specific agents have uniform or similar diffusion rates in the tissue (e.g., within 10% of each other, alternatively within 5%, 2%, 1%, or 0.1%).

[0047] The targeted and non-targeted agents each comprise a fluorophore. Fluorophores (or fluorescent dyes) absorb light of an excitation wavelength(s) and then emits that light at slightly longer wavelength(s). This allows fluorophores to act as labels in various assays, and they can be used to identify, quantify, and visualize molecules, cells, and other targets. Examples offluorophores include fluorescein dyes (e.g., fluorescein, fluorescein isothiocyanate, naphthofluorescein, etc.), cyanine derivatives (e.g., carbocyanine, merocyanine, CY-3, CY-5, CY-3.5, CY-5.5, etc.), rhodamine dyes (e.g., carboxytetramethyl-rhodamine, carboxyrhodamine 6G, carboxy-X-rhodamine, lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine, etc.), coumarin dyes (e.g., coumarin, methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin, etc.), and others. Fluorescent dyes are commercially available from many sources such as Thermo Fisher Scientific (Waltham, MA) and LICORbio (Lincoln, NE), including those marketed as ALEXA FLUOR® dyes, OREGON GREEN® dyes, IRDye® dyes, BODIPY® dyes, and / or DYLIGHT® dyes, for example, ALEXA FLUOR 350, ALEXA FLUOR 488, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 633, ALEXA FLUOR 660, ALEXA FLUOR 680, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, IRDye 680, IRDye 700, IRDye 750, IRDye 800, etc. In some embodiments, the fluorophore is a NIR fluorescent dye which refers to fluorophores having emission wavelengths from about 560 nm to 860 nm.

[0048] In some embodiments, each of the targeted fluorescent agents can comprise a fluorophore selected from the group consisting of optical dyes, such as OregonFluor 550 (OF550), OregonFluor 650 (OF650), IRDye 680LT (IR680), Alexa Fluor 700 (AF700), Alexa Fluor 750 (AF750), and IRDye 800CW (IR800). In some embodiments, at least one of the targeted fluorescent agents comprises OregonFluor 550 (OF550), or OregonFluor 650 (OF650), or IRDye 680LT (IR680), or Alexa Fluor 700 (AF700), or Alexa Fluor 750 (AF750). In some embodiments, a non-targeted fluorescent agent comprises IRDye 800CW (IR800).

[0049] In some embodiment of the present methods, a set of targeted fluorescent agents is used. The set of targeted fluorescent agents can comprise 3, 4, 5, 6 or more targeted fluorescent agents, each with a different fluorophore and a binding agent that specifically binds a different protein. The methods comprise obtaining or determining reference fluorescence emission data for each of the targeted fluorescent agents and for a non-targeted fluorescent agent. The reference fluorescence emission data can be determined for each of the targeted fluorescent agents and for the non-targeted fluorescent agent in a biologically relevant solvent, such as alipid emulsion. The reference fluorescence emission data can be used for spectral unmixing when the fluorescence emission data is acquired as multispectral data.Illuminating The Tissue & Acquiring Emission Wavelength Data

[0050] Tissue can be illuminated one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and by a non-targeted fluorescent agent when present.

[0051] In some embodiments, a sample is illuminated with a broadband lamp filtered through a multiband excitation filter at stimulus wavelength(s) in bands or ranges of from about 535 to about 545 nm, from about 620 to about 645 nm, and from about 740 to about 750 nm. A multiband excitation filter can comprise passbands of from about 538 to about 543 nm, from about 623 to about 641 nm, and from about 743 to about 747 nm.

[0052] Targeted fluorescence emission data (as well as non-targeted fluorescence emission data) can be acquired for one or more emission wavelengths associated with the first targeted fluorescent agent. In some embodiments, the targeted fluorescence emission data and the nontargeted fluorescence emission data are acquired by channels of a multichannel photodetector.

[0053] In some embodiments, the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired together, and the method further comprises spectral unmixing of the targeted and non-targeted fluorescence emission data.

[0054] The fluorescence emission data can be acquired through a multiband emission filter to obtain narrowband images in 5 nm increments from about 560 to about 860 nm. In some embodiments, the fluorescence emission data is acquired through a liquid crystal tunable filter. Alternatively, the fluorescence emission data can be acquired in bands or ranges of from about 555 to about 610 nm, from about 655 to about 730 nm, and from about 760 to about 850 nm. Alternatively, the fluorescence emission data can be acquired in bands or ranges of from about 557 to about 602, from about 660 to about 722, and from about 768 to about 846 nm.

[0055] In some embodiments, the present methods also comprise acquiring autofluorescence emission data prior to contacting the sample with the set of targeted fluorescent agents and the non-targeted fluorescent agent, and correcting the targeted fluorescence emission data using the autofluorescence emission data.

[0056] Imaging systems are commercially available which can illuminate a sample and acquire emissions wavelength data. In some embodiments, the Maestro imaging system is used forilluminating the tissue and acquiring fluorescence emission data. The Maestro M-MSI-500-FLEX imaging system can accommodate any combination of excitation and emission filters in the 500 to 900 nm wavelength range to suit experimental parameters. The stage can also be adjusted to bring the sample closer to the camera’s field of view, and the lighting can be tuned for even illumination at the desired stage height. Other notable hardware components of the Maestro imaging system includes a solid-state liquid crystal tunable filter, and a high-resolution CCD imaging sensor.

[0057] In some embodiments four fluorescent dyes are used to distinguish individual concentrations of PDL1, PD1, and CD80. These fluorescent dyes are advantageous due to minimal spectral overlap between their peak emission wavelengths of the dyes (>20 nm separation between peaks). Further, one can use a single multi-band excitation filter (passbands: 538-543, 623-641, 743-747nm) to excite all four fluorophores and a single multi-band emission filter (passbands: 557-602, 660-722, 768-846 nm) to collect emission (as illustrated in FIG. 3 panels (a-b)). The fluorophores IRD680, AF750, and IRD800 have also been successfully tested for PAI in both in vivo and ex vivo excised tissue samples. Minor spectral sensitivity was observed between IRD800 to the other fluorophores, but the percent difference between IRD800 alone or in a mixture with other dyes was within an acceptable range that minimally impacts the final results (as illustrated in FIG. 3 panel (c)). To confirm, the BPRPAM for each receptor was compared when the signal from the untargeted curves in the tumor-mimicking phantoms was decreased by 14%. Assuming that the overestimation of IRD800 is constant, the BPRPAM values did not change when mPAIR was run using recovered versus expected untargeted signals. Further studies to investigate the effects of IRD800 in a multi-fluorophore mixture over a range of concentrations may determine whether its degree of overestimation using mPAIR stays constant. Overall, clear spectral distinction was achieved within the panel of dyes to ensure accurate identification and quantification of each receptor in the PDL1 axis, supporting the fluorophore selection and spectral unmixing method used in mPAIR.

[0058] In some embodiments, the selections of fluorophores paired with binding agents and a non-specific agent can be selected based on concentrations observed in in vivo tumor models of the proteins to be targeted. By way of example, anti-PDLl, anti-PDl, and anti-CD80 antibodies can be conjugated to AF750, IRD680, and AF700, respectively. PDL1 is reported to be highly expressed in tumors and was thus paired with AF750 because its low quantum efficiency (0.12)avoids potential PDL1 signal saturation from tumors. Although IRD680 has a lower reported quantum yield (0.19) than AF700 (0.25), it was observed that IRD680 consistently produced a higher signal intensity than AF700 in the Maestro at lower concentrations (see FIG. 3 panel (c)). PD1 was thus paired with IRD680 due to its dynamic expression levels, which can start low but increase significantly as T cells become dysfunctional over tumor progression. The untargeted agent (IgG isotype control) was conjugated to IRD800 because IRD800 has the lowest quantum efficiency (0.07) among the dyes, making it desirable to use as the untargeted signal. Liquid phantom experiments also showed that IRD800 is the most challenging regarding signal accuracy in a four-dye mixture, further justifying its use for background correction.Spectral Unmixing

[0059] Multi-spectral imaging can capture spectrally resolved information at a defined wavelength range for each pixel in an image. Liquid crystal tunable filters (LCTFs) are utilized to permit a narrow bandpass of light at specific wavelengths to pass through and enable the generation of an image cube - a series of images taken at the specified wavelength interval. The three-dimensional data within an image cube, including x, y, and L, is used to characterize the combined spectra of autofluorescence and fluorophore labels used. A library of reference basis spectra for autofluorescence and fluorophores is then utilized in a spectral unmixing algorithm to separate the contributions of each fluorophore and autofluorescence and generate spectral curves for each component. Alternatively, autofluorescence can be directly subtracted from the image cube. The result is a set of images representing the isolated fluorescence signals from the fluorophore labels used and / or autofluorescence contributing to the image.

[0060] In some embodiments, NIR fluorophores are used to combat challenges in in vivo optical imaging. Tissue autofluorescence is a significant source of ubiquitous signals that can limit fluorophore detection and accuracy. It results from various endogenous sources and varies across tumor subtypes, further adding complexity to the accurate interpretation of fluorescence imaging results. Generally, tissue autofluorescence is prominent in the visible region while minimal in the longer, NIR wavelength regions of the electromagnetic spectrum.

[0061] Additional detail regarding spectral unmixing or fluorescence detection can be found in Tichauer et al. US Pat. No. 11,564,639; Determan et al. US Pat. No. 8,462,981; Levenson et al. US Pat. No. 8,639,043; Levenson et al. US Pat. No. 8,879,812; Bamford et al. US Pat. No.9,792,693; Chukka et al. US Pat. No. 9,996,924; Cooper US Pat. No. 10,753,875; and Xu et al. US Pat. No. 11,730,370, each of which is incorporated by reference herein.

[0062] In some embodiments of the present methods, the spectral unmixing comprises representing the targeted fluorescence emission data and the non-targeted fluorescence emission data as a linear combination of each of the fluorescent agents. A least-squares fitting can be performed to calculate relative contributions of each of the fluorescent agents.

[0063] In some embodiments, the step of acquiring fluorescence emission data comprises acquiring a set of multispectral images of the sample, wherein each of the multispectral images corresponds to a different wavelength band or range.

[0064] In some embodiments, a three-dimensional (m x n x 1) image cube is generated from each acquisition of fluorescence emission data, where m is the number of row-pixels, n is the number of column-pixels, and 1 is the number of wavelengths. A measured signal intensity ^(A) at wavelength for each of the fluorescent agents at each pixel of each of the multispectral images can be determined by the formula:where x is the relevant amount of the fluorescent agent z, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent.Multi-spectral paired-agent imaging (PAI) of receptors (mPAIR)

[0065] The present disclosure also provides methods for detecting or quantifying two or more biomarkers (such as a cellular receptors) in the tissue simultaneously by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR). The mPAIR methods expand upon the methods and steps as described above, in that the foregoing targeted fluorescent agent is a first targeted fluorescent agent, and the mPAIR methods further comprise contacting the tissue with a second targeted fluorescent agent, illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent; acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; and correcting the second targeted fluorescence emission data using the non-targeted fluorescence data.

[0066] For example, the mPAIR methods can comprise detecting or quantifying PD-1 or CD80 in the tissue simultaneously with PD-L1 (or with a-PD-Ll and / or f-PD-Ll) by paired agent imaging. In some embodiments, the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80). In some embodiments, the methods comprise detecting or quantifying a receptor in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR). For example, the methods can comprise detecting or quantifying PD-1 or CD80 in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by mPAIR.

[0067] The present methods can comprise detecting or quantifying one or more biomarkers in tissue of the subject relevant to immunotherapy response, such as PD-L1, a-PD-Ll, f-PD-Ll, PD1, and / or CD80. The biomarker(s) are detected or quantified based on fluorescence emission data from a targeted fluorescent agent(s) that binds to the biomarker(s). In some embodiments, the targeted fluorescent agent binds to PD-L1. In some embodiments, the targeted fluorescent agent that binds to a-PD-Ll and / or f-PD-Ll and does not specifically bind to PD-L1 complexed with PD-1 or CD80. The methods can comprise contacting the tissue with a PD-l-targeted fluorescent agent and a CD80-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents; acquiring PD-1 -emission data and CD80-emission data for one or more emissions wavelengths associated with the a PD-1- and CD80-targeted fluorescent agents; correcting the PD-l-emission data and CD80-emission data using the non-targeted fluorescence data; and detecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

[0068] The present methods can also comprise providing a spatial and / or temporal image of PD-L1, a-PD-Ll and / or f-PD-Ll in the tissue, and / or providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue. FIG. 5 panel (d) and FIG. 13 panel (c) show spatial images of PD-L1, PD-1 and CD80 in tissues.Correcting Emission Data

[0069] In some embodiments, the targeted fluorescence emission data is corrected by subtracting and normalizing by the non-targeted fluorescence data. In some embodiments, the targeted fluorescence emission data is corrected by using a ratiometric calculation. The ratiometric calculation can comprise dividing the targeted fluorescence emission at one or moretime points by an intensity corrected untargeted fluorescence emission at the same one or more time point minus one. The intensity corrected untargeted fluorescence emission can be determined by multiplying the untargeted fluorescence emission by a ratio of targeted fluorescence emission to untargeted fluorescence emission collected from (a) a standard without the biomarker of interest at the same time point, or (b) from an earlier time point where concentrations of the targeted fluorescent agents and the non-targeted fluorescent agents are essentially the same. In some embodiments, the targeted fluorescence emission data is corrected by fitting sequences of normalized targeted and untargeted fluorescence emission intensities with a nonlinear least-squares algorithm.Rinsing paired-agent kinetic model (RPAM)

[0070] The present disclosure also provides for calculating a biomarker binding potential of the tissue using a rinsing paired-agent kinetic model (RPAM). RPAM was previously established as a mathematical model to calculate a single BP (BPRPAM) from a sequence of targeted and untargeted imaging agent signals with repeated rinsing. Xu, X., et al., Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues. Phys. Med. Biol. 62, 5098-5113 (2017).

[0071] RPAM is adapted for the present methods to calculate BPRPAM values for multiple agents and used experimentally validated diffusion coefficients of each to determine their appropriate incubation time with our phantom. In brief, a background image cube is acquired, and then incubated simultaneously with all four imaging agents. An initial image cube was acquired to represent the first time point. After rinsing the model with PBS, subsequent image cubes were captured to assess tissue clearance dynamics Image cubes were spectrally unmixed using the reference spectral library (characteristic spectra of “pure” fluorophore) created from the liquid phantom experiments, and the autofluorescence spectra from the autofluorescence acquisition. BPRPAM for each targeted receptor was determined using a nonlinear least-squares fit of:Equation 4:where ST(t) is the sequence of targeted image agent signals; Sc(t) is the sequence of the untargeted imaging agent signals; ttis any single imaging time point post rinsing (the second post-rinse image was used for BPRPAM); and t is a time vector. In some embodiments, themethods comprise calculating a PD1 binding potential and or a CD80 binding potential using the RPAM.Tissues

[0072] The present methods comprise contacting a tissue with a targeted fluorescent agent. The tissue can comprise a sample extracted from a subject, a formalin-fixed paraffin-embedded (FFPE) sample, or a fresh frozen sample. In some embodiments, the tissue is tumor tissue and tumor-draining lymph node (TDLN) tissue. The tissue can be in vivo. For instance, the tissue can be present in a living subject, and a targeted fluorescent agent and optionally a non-targeted fluorescent agent are administered to the subject. As an example, a targeted fluorescent agent and optionally a non-targeted fluorescent agent are administered to the subject for a surgical procedure on the tissue. In some embodiments, a biomarker binding potential, concentration or location is dynamically assessed in the tissue in real-time.Diagnostic Methods

[0073] The present methods can also comprise determining a biomarker score (such as a PD-L1 score, an a-PD-Ll score and / or a f-PD-Ll score) for the tissue. In some embodiments, the methods also comprise determining a biomarker binding potential (such as a PD-L1 binding potential) of the tissue. In some embodiments, the a-PD-Ll score and / or the f-PD-Ll score is determined from the PD-L1 binding potential.

[0074] The methods can also comprise comparing the PD-L1 score, the a-PD-Ll and / or the f-PD-L1 score to a PD-L1 threshold value, an a-PD-Ll threshold value and / or a f-PD-Ll threshold value, respectively, and identifying the subject as a likely responder if the PD-L1 score, the a-PD-Ll score and / or the f-PD-Ll score is greater than or equal to the PD-L1 threshold value, the a-PD-Ll threshold value and / or the f-PD-Ll threshold value, respectively, and / or identifying the subject as a likely non-responder if the PD-L1 score, the a-PD-Ll score and / or the f-PD-Ll score is below the PD-L1 threshold value, the a-PD-Ll and / or f-PD-Ll threshold value, respectively. The methods can also comprise maintaining or adjusting administration of the immunotherapy to the subject based on the PD-L1 score, the a-PD-Ll score and / or the f-PD-Ll score.

[0075] The present methods can also comprise monitoring treatment of a patient undergoing a therapy that increases or decreases PD-L1, a-PD-Ll and / or f-PD-Ll. For instance, the methods can comprise monitoring treatment of a patient undergoing anti-PD-1 therapy and / or anti-PD-Lltherapy and / or anti-CD80 therapy. The subject can be administered an immunotherapy to the subject before and / or after detecting or quantifying PD-L1, a-PD-Ll and / or f-PD-Ll. Examples of immunotherapy that may increase or decrease PD-L1, a-PD-Ll and / or f-PD-Ll include anti-PD-1 antibodies such as nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, and tislelizumab; anti-PD-Ll antibodies such as atezolizumab, durvalumab, avelumab, envafolimab, and sugemalimab; anti-CD80 antibodies; and other agents that bind to one or more PD-L1 axis proteins.

[0076] The present methods can also comprise monitoring treatment of the subject with the immunotherapy. In some embodiments, the subject is identified as a responder if the PD-L1 score, the a-PD-Ll and / or f-PD-Ll score is greater than or equal to a PD-L1 threshold value, an a-PD-Ll threshold value and / or a f-PD-Ll threshold value, respectively, and / or identifying the subject as a non-responder if the PD-L1 score, the a-PD-Ll score and / or the f-PD-Ll score is below the PD-L1 threshold value, the a-PD-Ll threshold value and / or the f-PD-Ll threshold value, respectively. In some embodiments, the methods comprise maintaining or adjusting administration of the immunotherapy to the subject based on the PD-L1 score, the a-PD-Ll score and / or the f-PD-Ll score.

[0077] The present methods can also comprise obtaining a sample comprising tissue from the subject, and contacting the sample with a targeted fluorescent agent and a non-targeted fluorescent agent. In some embodiments, the sample comprises melanocytes and / or blood.

[0078] In some embodiments, the present methods are used with a subject having adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma, ependymoma, breast cancer, carcinoid tumor, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell and small cell, lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma and other plasma cell neoplasms, mycosisfungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, or Wilms' tumor. In some embodiments, the subject has melanoma. In some embodiments, the subject has T-cell lymphoma.Detecting or quantifying a-PD-Ll and / or f-PD-Ll

[0079] The present methods can also comprise detecting or quantifying a-PD-Ll and / or f-PD-Ll in the tissue based on the targeted fluorescence emission data. In some embodiments, cell-associated concentration of a-PD-Ll and / or f-PD-Ll in the tissue is quantified.

[0080] In some embodiments, the methods also comprise displaying a visual image of the tissue comprising a spatial representation of one or more biomarkers present in the tissue.

[0081] In some embodiments, the methods also comprise obtaining a white-light image of the tissue, and superimposing the visual image on the white-light image.

[0082] In some embodiments, the methods also comprise generating a biomarker binding potential map of the sample.

[0083] In the present methods, imaging all three PD-L1 axis proteins can provide crucial context because PDL1 expression alone may not fully reflect functional binding capacity. For example, PDL1 interactions with CD80 can impede its binding to PDL1, suggesting that PDL1 must be both present and functionally accessible for anti-PDl therapies to work effectively. Zhang et al., Blockade of trans PD-L1 interaction with CD80 augments antitumor immunity. Proc. Natl. Acad. Set. U.S.A. 120, e2205085120 (2023); Zhao et al., PD-LLCD80 CisHeterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways. Immunity 51, 1059-1073. e9 (2019); Sugiura et al., Restriction of PD-1 function by cis-PD-Ll / CD80 interactions is required for optimal T cell responses. Science 364, 558-566 (2019). Others has opted for a multiplexed fluorescence molecular imaging (FMI) strategy using NTR-II-b rare-earth nanoparticles to image PDL1 and CD8 in tumor-bearing micetreated with ICIs. Zhong et al., Nat Biotechnol 37 , 1322-1331 (2019). By visualizing these two proteins simultaneously in vivo, they observed favorable therapeutic responses to anti-PDLl therapy when high levels of PDL1 and CD8+immune cells were present at the tumor site, demonstrating the benefits of multiplexed imaging to monitor immunotherapy responses. While multiplexed FMI overcomes the limitations of single protein detection, it still faces challenges common to single-agent molecular imaging. Specifically, single-agent molecular imaging (irrespective of modality) can only report relative protein expression and cannot quantify accurate receptor concentrations, limiting their ability to fully characterize immune checkpoint availability. More importantly, targeted imaging agents can also accumulate non-specifically in the TME due to inherent properties of complex tumor biology; thus in oncology, imaging agent contrast only loosely tracks receptor expression. Tichauer et al., Mol Imaging Biol 14, 584-592 (2012); Aerts et al., Journal of N clear Medicine 50, 123-131 (2009); McLarty et al., Eur J Nucl Med Mol Imaging 36, 81-93 (2009)

[0084] The present mPAIR methods offer the advantage of incorporating a non-targeted agent, effectively eliminating background signal and allowing for quantitative measurements. As a result, mPAIR addresses a significant gap within current biomarkers for ICI therapy in future in vivo studies by providing a way to quantify the number of receptors that are functionally accessible for binding.

[0085] Although the present disclosure demonstrates mPAIR in a tumor-mimicking phantom model, limitations of using fluorophores within a narrow spectral window should be considered. IRD680 and AF700 were spectrally unmixed with high accuracy using a custom multi-band filter set, but IRD800 presented a few challenges as previously addressed. Specifically, IRD800’s signal appeared to increase when in a mixture, likely due to spectral overlap with AF750. This led to an unexpected reduction in AF750’s signal, which may be attributed to reabsorption effects where IRD800 effectively borrows some of the emitted photons from AF750 due to their close spectral proximity. This effect is likely minimal in the RPAM model due to the nonlinear fit of the dynamic behavior of the fluorophore rather than the STP BP method, where this could really affect results. These findings highlight the need to carefully consider interactions among fluorophores in a mixture, particularly between IRD800 and AF750, as we progress towards testing mPAIR in in vivo models. Another limitation of our study is using E.G7-OVA cells to establish the phantom model (FIGs. 1, 2). Although E.G7-OVA cells were intentionally selected to validate mPAIR’s ability to detect and quantify the PDL1 axis proteins simultaneously, TMEs are far more heterogeneous and can pose significant challenges. Variations in tissue optical properties, receptor densities, and diverse immune cell populations could influence mPAIR accuracy and imaging sensitivity. Despite these limitations, successfully validating mPAIR in preliminary tumor-mimicking phantom studies is a critical validation step before progressing to in vivo applications. Our next steps will be to test mPAIR in syngeneic murine tumor models to validate its performance in a setting with dynamic tumor-immune interactions.

[0086] The mPAIR methods described by the present disclosure could significantly expand the ability to select subjects who will or are likely to respond to ICI therapy and minimize unnecessary treatments. In some embodiments, the methods are used for quantifying immune checkpoint availability in real time. In this study, mPAIR was developed to interrogate the availability of the PDL1 axis proteins for anti-PDl treatment. However, mPAIR can be adapted to monitor other clinically relevant immune checkpoints in future studies, potentially expanding its use across various immunotherapy applications. Unlike static PDL1 biopsy measurements, mPAIR has the unique potential to capture the spatial and temporal fluctuations that may impact therapeutic response by quantifying receptor availability directly within the TME. Assessing the number of receptors available to bind to ICIs could better inform clinicians on the likelihood of their patients responding to treatment.EXAMPLES

[0087] The following methods and materials were used in the examples which follow (unless indicated otherwise in the individual examples).Cell culture

[0088] E.G7-OVA cells, a murine lymphoma cell line, were purchased from ATCC (Manassas, VA) and maintained in RPMI 1640 medium (Corning Incorporated, Coming, NY) supplemented with 10% fetal bovine serum (v / v, Cytiva, Marlborough, MA) and 1% penicillinstreptomycin (v / v, Corning Incorporated). Cells were cultured at 37°C in a 5% CO2 atmosphere.Brightfield and multi-spectral image collection

[0089] Brightfield images of E.G7-OVA tumor-mimicking phantoms were captured using the Spotimage camera (Diagnostic Instruments Inc, Sterling Heights, MI) on a Nikon Eclipse Ts2 inverted microscope (Nikon Instruments, Melville, NY). Images were viewed and exported directly using SPOT software (version 5.6, Spotimage, Sterling Heights, MI).

[0090] Multispectral images were collected using the Maestro M-MSI-500-FLEX imaging system. Illumination from a broadband 300 W Xenon lamp was filtered through a multiband excitation filter (passbands: 538-543, 623-641, 743-747 nm), and fluorescence signal was collected using a multiband emission filter (passbands: 557-602, 660-722, 768-846 nm) in conjunction with the system’s liquid crystal tunable filter to obtain narrowband images in 5 nm increments from 560 to 860 nm. The multiband excitation and emission filter sets were customized to accommodate the desired passband wavelengths (Chroma, Bellows Falls, VT). Each acquisition resulted in a three-dimensional (m x n x Z) image cube, where m is the number of row-pixels, n is the number of column-pixels, and I is the number of wavelengths -equivalent to the number of images. Background (pre-fluorophore administration) image were collected to obtain the autofluorescence basis spectra that was used in the spectral unmixing algorithm.Statistical methods

[0091] Unless otherwise indicated, all statistics were performed using Prism (version 10.0.3, GraphPad Software, Boston, MA) and Excel software (version 16.92, Microsoft, Redmond, WA). Dissociation constants for each imaging agent were determined using the one-site specific binding model on saturation curves. Unpaired two-tailed t-tests were used to compare the mean dissociation constants between two imaging agents, signal intensity between NIR dye alone and in combination with other dyes in a liquid phantom, and diffusion coefficients between two imaging agents. A one-way ANOVA test was used to compare PDL1, PD1, and CD80 receptors on E.G7-OVA cells. Pearson correlation, Lin’s concordance correlation coefficient (CCC), bias correction factor, and root mean squared error (RMSE) were calculated between log-transformed PDL1, PD1, and CD80 receptors measured by flow cytometry and those quantified in E.G7-OVA tumor-mimicking phantoms. Spearman correlation was calculated between PDL1, PD1, and CD80 receptors measured by flow cytometry and those quantified in E.G7OVA tumors. P < 0.05 was considered statistically significant for all experiments.Example 1

[0092] In this example, the binding affinities of targeted fluorescent agents for their respective PDL1 axis proteins (PDL1, PD1, and CD80) were determined, and the numbers of PDL1, PD1, and CD80 receptors on a selected cell line were evaluated. PAI imaging is based on the condition that the untargeted agent has similar pharmacokinetic and physical properties to the single targeted agent to correct for non-specific binding of the targeted agent. For the development of mPAIR, the recovered BP signal is dependent on both the receptor concentration and binding affinity of the antibody-receptor pair. Thus, in order to accurately compare receptor concentrations, the binding affinity, or the dissociation constant, must be determined. Anti-PDl (BioLegend, San Diego, CA), anti-PDLl (Invitrogen, Waltham, MA), and anti-CD80 antibodies (BioLegend) were selected and labelled with Alexa Fluor 488 (AF488) NHS ester (Invitrogen) for saturation binding and quantitative flow cytometry experiments. All antibodies targeting the PDL1 axis proteins were specific to mouse, while the untargeted antibody was specific to rat. To ensure similar binding and diffusion properties, all antibodies selected for the study were of the IgG2aK clonal isotype.

[0093] 200,000 cells were distributed in a FACS buffer solution, comprised ofDPBS (Coming Incorporated), 3mM EDTA (Fisher Chemical, Pittsburgh, PA), 2.5% FBS, and murine Fc block (Bio X Cell, Lebanon, NH). Anti-PDl -AF488 (0.005 - 37.5 nM), anti-PDLl -AF488 (0.005 -37.5 nM), and anti-CD80-AF488 (0.039 - 300 nM) antibodies were added to E.G7-OVA cells in separate 1 :2 serial dilution conditions. Cells were stained in the dark at 4°C for 30 minutes before washing three times with PBS. Cells were resuspended in FACS buffer (without murine Fc block) and 7-AAD dye (Invitrogen, Waltham, MA) was added to exclude dead cells from analysis. Flow cytometry data of stained samples were acquired using the Cytek Aurora (Cytek Biosciences, Fremont, CA) and analyzed with FlowJo software (version 10.8, Ashland, OR). Three independent trials of the study were performed.

[0094] All three targeted fluorescent agents reached saturation at 10 nM, with dissociation constants (Xd) of 0.34 ± 0.07 nM for PDL1, 0.30 ± 0.2 nM for PD1, and 0.38 ± 0.2 nM for CD80 (FIG. 1). The binding affinities of the selected antibodies indicated they were suitable for use as targeted agents in the present methods, with similar binding affinities for their respective PDL1 axis proteins.

[0095] Standard curves for AF488 fluorescence quantification were generated using the Quantum AF488 Molecules of Equivalent Soluble Fluorochrome (MESF) microsphere kit (Bangs Laboratories, Fishers, IN). Antibodies (PD1-AF488, PDL1-AF488, and CD80-AF488) were used as targeted fluorescent agents at concentrations sufficient to achieve receptor saturation at 10 nM, as determined from the foregoing binding studies. The fluorescence intensities of stained samples were converted to MESF units using the QuickCal template (version 3.0) provided by Bangs Laboratories to determine the number of fluorophores bound per cell. Receptor number was calculated by dividing the number of fluorophores per cell by the number of AF488 fluorophores per antibody molecule, as measured by absorption spectroscopy using the NanoDrop.

[0096] E.G7-OVA cells were evaluated as a suitable model for quantifying concentrations of PDL1 axis receptors. The number of PDL1, PD1, and CD80 receptors in E.G7-OVA cells were measured with the targeted fluorescent agents using QFC (FIG. 2(a)). The expression of PDL1 and PD1 was measured across the whole E.G7-OVA population because a continuous distribution of these receptors was observed among the cells. CD80 expression, however, was present in a sub -population of E.G7-OVA cells, allowing for a clear distinction between receptor-positive and receptor-negative cells. One-way ANOVA showed significant differences in the expression of PDL1, PD1, and CD80 within the E.G7-OVA cell population, with an average of 1.27 x 103± 0.41 x 103PDL1 receptors and 1.05 x 104± 0.41 x 103PD1 receptors per cell across the entire population, and 8.90 x 102± 0.49 x 103CD80 receptors per receptorpositive cell (FIG. 2(b)).

[0097] Anti-PDLl, anti-PDl, and anti-CD80 antibodies of the same clonal isotype were selected as targeted imaging agents. The imaging agents exhibited similar Kdvalues ranging from 0.30 nM to 0.38 nM and reached saturation at 10 nM, indicating comparable binding characteristics (FIG. 1).Example 2

[0098] In this example, targeted fluorescent agents for distinguishing PDL1 axis proteins were prepared. The anti-PDl, anti-PDLl and anti-CD80 antibodies of Example 1 were conjugated with the following dyes: anti-PDl with IRDye 680LT (IRD680) NHS ester (LLCOR Biosciences, Lincoln NE), anti-CD80 with Alexa Fluor 700 (AF700) NHS ester (Invitrogen),anti-PDLl with Alexa Fluor 750 (AF750) NHS ester (Invitrogen), and rat IgG a (IgG) isotype control (Leinco Technologies, St. Louis, MO) with IRDye 800CW NHS ester (LLCOR Biosciences). The antibodies were prepared for labeling as described by the manufacturer’s instructions. Pierce Zeba desalting chromatography columns (7K MWCO, Thermo Scientific, Waltham, MA) were used to separate labelled antibodies from free dye. Absorption spectra were taken of all samples using the NanoDrop (Thermo Scientific) to ensure that the conjugated antibodies had a dye to protein ratio of three or less (FIG. 6).Example 3

[0099] A liquid phantom model was developed for use in evaluating targeted fluorescent agents. The model consisted of the fluorophore(s) diluted in PBS (Coming Incorporated), 1% bovine serum albumin (BSA, VWR Life Science, Radnor, PA) to mimic tissue solubility, and 1% intralipid (Fresenius Kabi, Uppsala, Sweden) to account for tissue scattering. Dye concentrations were adjusted to achieve similar emission intensities across the different fluorophores. Phantoms were imaged using the Maestro M-MSI-500-FLEX imaging system (Cambridge Research & Instrumentation Inc., CRi) in 96-well black plates with optical bottoms (Thermo Scientific).Example 4

[0100] In this example, a medium for use in a tumor-mimicking phantom model was developed, and it was demonstrated that two-dimensional mean squared displacement determines diffusion coefficient of targeted fluorescent agents that target the PDL1 axis.

[0101] Agarose powder (Fisher BioReagents, Pittsburgh, PA) was first dissolved in PBS to form a 0.6% agarose mixture (w / v) and then cooled to 39°C while stirring to ensure a homogenous mixture before mixing it 1 : 1 with warmed PBS to create a 0.3% agarose mixture (w / v). The mixture was uniformly distributed into each well of a custom 3D-printed four- well plate, with a diameter of 35.34 mm and a depth of 19.10 mm. The wells were kept in a digital dry bath for 15 to 30 minutes so that the agar did not prematurely gel. A hole was then made at the center of the agarose in each well using a 16-gauge x %" Luer stub needle to accommodate a 12 pL solution of each fluorescent antibody (IRD680-PD1, AF700-CD80, AF750-PDL1, and IRD800-IgG) for imaging. For each experiment, one hole per well was used to collect data.

[0102] A background image of the medium was captured using the Maestro imaging system. Targeted fluorescent agents were added to the agar surface and allowed to penetrate for 120 minutes. Excess solution remaining on agar surface was carefully removed, and rinsed with PBS for 5 minutes. Post-rinse, excess PBS was removed, and phantom image was captured. The rinse and imaging cycle was repeated at least 10 times.

[0103] The Pearl Impulse Small Animal Imaging System (LICORbio) was used to collect images at a set resolution of 170 pm. An image of the agarose in the well was captured to serve as a background before adding 12 pL of a 1000 nM antibody-dye solution and initiation of an image time series where the system was set to capture images at 10-minute intervals for 60 minutes. The images were exported as TIF fdes for analysis in MATLAB. We focused on analyzing images from the 700 nm channel for IRD680-PD1 and AF700-CD80 and the 800 nm channel for AF750-PDL1 and IRD800-IgG.

[0104] The image scale was determined by taking a white light image of a transparent ruler at the same resolution and determining that 1 cm corresponds to 59 pixels using MATLAB. Using the second acquisition, a rectangular region-of-interest (ROI) was drawn around the hole to crop the image to the area of focus and avoid bright spots at the well edges due to light scattering. This ROI was saved and applied to all subsequent acquisitions for consistency. For each image, pixel values were summed along the columns to produce a 1 -dimensional intensity profile. Since these profiles followed a normal distribution, the full width at half maximum of each curve was calculated to estimate the distance the dye diffused from the center hole at each time step. Occasionally, the holes made using the Luer stub needle were irregular. To address this, each acquisition was analyzed by rotating the image through 0°, 45°, 90°, and 135°. The following diffusion coefficient and experimental time calculations were performed for each image rotation, and the mean across all four ± standard deviation is reported. The movement of the fluorescent antibody through the tissue-mimicking agar was modeled using the two-dimensional mean squared displacement for simple Brownian diffusion:Equation 1: {r2) = 4Dtwhere D is the diffusion coefficient (cm2 / s) and t is the time of image acquisition (s). A best-fit line was determined from the plot of the squared radius versus time and the diffusion coefficient was determined by dividing the slope by 4 according to Equation 1.The experimental time (texp) required for the fluorescent antibody to distribute evenly throughout the tissue mimicking phantom was determined from using the two-dimensional mean squared displacement equation but using the depth of the tissue mimicking phantom (x = 0.106 cm) and the experimentally determined diffusion coefficient ( ):Equation 2:

[0105] Imaging agents labeled with Alexa Fluor dyes demonstrated significantly higher diffusion coefficients (PDL1-AF750: 8.35 X 10'7± 0.26 X 10'9cm2 / s and CD80-AF700: 9.00 X10’7± 0.27 x 10‘9cm2 / s) compared to IRDye dyes (PD1-IRD680: 5.47 X10’7± 0.48 X 10'9cm2 / s and IgG-IRD800: 4.93 X10’7± 0.77 X IO’9cm2 / s).Example 5

[0106] In this example, the medium of Example 3 was used with E.G7-OVA cells to make a tumor-mimicking phantom model. To accurately validate mPAIR in a tumor phantom, the imaging agents were evaluated for ability to reach their target receptors within a 3D cell model. E.G7-OVA cells were seeded in 0.3% solid agarose gel to a glandular tumor-mimicking phantom model. Agarose powder was first dissolved in PBS to form a 0.6% agarose mixture and then cooled to 39°C while stirring to ensure a homogenous mixture. Simultaneously, cells were prepared by resuspending 30 million E.G7-OVA cells in 500 L of PBS. This cell suspension was mixed with 500 / zL of the 0.6% agarose solution to create a final 0.3% agarose mixture. The resulting mixture was transferred to a 6-well glass bottom plate (Cellvis, Mountain View, CA) and allowed to solidify for five to ten minutes. Phantoms without any cells served as the control. Three independent trials were performed.

[0107] The thickness of the E.G7-OVA 3D tumor phantom was measured to be 10.6 mm; thus, the estimated time it would take for each targeted fluorescent agent to diffuse through the agar layer was 56.6 ± 1.8 minutes for PDL1-AF750, 86.2 ± 7.8 minutes for PD1-IRD680, 55.3 ± 1.4 minutes for CD80-AF700, and 99.7 ± 14 minutes for IgG-IRD800 (FIG. 4 panels (a-b)).Therefore, to allow sufficient time for all imaging agents in a mixture to engage with their respective receptor, the tumor phantoms were incubated with the targeted fluorescent agents for 120 minutes (FIG. 4 panel (c)).

[0108] The diffusion coefficients of each targeted and untargeted agent were measured to determine how long it would take to penetrate the solidified agar layer embedded with E.G7-OVA cells. Through experimental validation of diffusion rates, significant differences were observed in diffusion coefficients between dyes from different families (i.e., Alexa Fluor versus IRDye dyes) despite being conjugated to antibodies with similar molecular sizes. Notably, the dyes themselves also had comparable molecular weights (IRD680 and AF700: 1400 g / mol; AF750: 1300 g / mol; IRD800: 1200 g / mol), suggesting that variations in chemical properties may account for the varying degrees of molecular “stickiness”. These findings overall emphasize the importance of experimentally validating diffusion rates to provide agents that diffuse uniformly for an appropriate amount of time.Example 6

[0109] In this example, the development of a multi-spectral unmixing algorithm is described. Spectral unmixing was conducted using the Maestro imaging system software (version 2.6, CRi). Basis spectra were necessary to build a reference spectral library for spectral unmixing. The spectral library for each fluorescent dye was first created from basis spectra obtained from image cubes of liquid phantoms with the corresponding fluorescent dye. Each curve should be a “pure” representation of the fluorophore’s emission. Background subtraction was performed to isolate the pure spectra of each fluorophore; that is, the intralipid plus BSA control spectra was subtracted from each fluorophore’s spectra. All basis spectra were then normalized to their respective maxima.

[0110] After defining the spectral library for each fluorescent dye and autofluorescence, image cubes were unmixed using either a 5-color spectral library (Cy5, IRD680, AF700, AF750, and IRD800), a 4-color spectral library (IRD680, AF700, AF750, and IRD800, or a 4-color plus autofluorescence spectral library (Cy5, IRD680, AF700, AF750, IRD800, and Intralipid + BSA only). In cases where autofluorescence subtraction was evaluated, the autofluorescence spectral library was used to subtract the intralipid and BSA spectrum from an image cube. Unmixed images were subsequently imported into FIJI (version 2.1.0, NIH, Bethesda, MD) for analysis, where fluorescence intensities were quantified.

[0111] Image cubes were loaded in MATLAB as three-dimensional arrays. Total signal can be represented as a linear combination of each component (individual fluorophores and background fluorescence). As such, a least-squares fitting approach was used to determine the relative contributions of each. This was achieved using the built-in MATLAB function Isqlin,where the square difference between measured multispectral data and previously determined basis spectra (i.e., characteristic emission spectra of each fluorophore and background signal) was minimized. The linear system was represented as:Equation 3:where y(2i) is the measured signal intensity at a pixel at wavelengthx is the coefficient of component i, for M different components that is output from the fitting procedure (i.e., the contribution of each at a given pixel); and F is the basis spectra for each component (fluorophores and autofluorescence). The fitting algorithm was constrained such that the coefficients cannot be negative. Spectral unmixing was performed on a pixel-by-pixel basis to reconstruct images of each component where signal intensity was proportional to its relative contribution. The five-color unmixing results in five different images - one each of IRD680, AF700, AF750, IRD800 and autofluorescence.Example 7

[0112] In this example, the use of targeted fluorescent agents for detecting PDL1 axis proteins by multi-spectral paired agent imaging of receptors (mPAIR) was evaluated. It was demonstrated that a panel of fluorophores can be accurately unmixed using a single excitation and emission filter set.

[0113] Fluorescence tissue imaging, including PAI, commonly utilize NIR probes for in vivo applications due to their ability to minimize autofluorescence and light scattering while enabling deeper tissue penetration. For mPAIR of three proteins, four NIR fluorophores were used to reliably and accurately be spectrally unmixed utilizing the Maestro imaging system. The fluorophores IRD680, AF700, AF750, and IRD800 were selected based on their known spectral distinction from one another (illustrated in FIG. 3 panel (a)). Five basis spectra - one each for IRD680, AF700, AF750, IRD800, and background (intralipid / BSA only) - were collected to perform spectral unmixing in dye combination studies (FIG. 3 panel (b)).

[0114] To test the accuracy of the unmixing algorithm to determine spectrally distinct fluorescence emission signals, IRD680, AF700, AF750, and IRD800 were tested alone and incombination with each other in a liquid phantom model. Each well contained a total volume of 300 pL per condition. Liquid Phantoms without any fluorophore served as the control.

[0115] In each case, the fluorescence signal for every condition was unmixed using the four-dye and intralipid / BSA spectral library.

[0116] As shown in (FIG. 3 panel (c), and FIG. 7). It was found that the spectral unmixing method for mPAIR successfully identified the fluorescent signal of IRD680, AF700, and AF750, both when the fluorophores were alone and in four-dye mixtures, with minimal crosstalk between the fluorophore spectral signals. However, a significant difference was observed in IRD800 emission when tested alone versus when it was part of the four-dye mixture (p < 0.0001 by unpaired two-tailed t-test). Specifically, there was a 14% increase in IRD800 emission when in a mixture with the other dyes (FIG. 3 panel (c), and FIG. 7).Example 8

[0117] In this example, it was demonstrated that mPAIR quantifies receptor concentrations of PDL1, PD1, and CD80 in a tumor-mimicking phantom model. The present methods were used to determine binding potentials using a rinsing paired-agent kinetic model (RPAM). RPAM was adapted in this example to calculate BPRPAM values for multiple agents. The experimentally validated diffusion coefficients of the agents (see Example 4) were to determine their appropriate incubation time with the tumor-mimicking phantom.

[0118] In brief, a background image cube of the E.G7-OVA tumor-mimicking phantom was acquired, and then incubated simultaneously with all four imaging agents for 120 minutes. An initial image cube was acquired to represent the first time point. After rinsing the phantom model with PBS, subsequent image cubes were captured to assess tissue clearance dynamics (FIG. 4(c)). Image cubes were spectrally unmixed using the reference spectral library (characteristic spectra of “pure” fluorophore) created from the liquid phantom experiments and the autofluorescence spectra from the background acquisition.

[0119] BPRPAM for each targeted receptor was determined using a nonlinear least-squares fit of Equation 4:where ST(t) is the sequence of targeted image agent signals; Sc(t) is the sequence of the untargeted imaging agent signals; ttis any single imaging time point post rinsing (the second post-rinse image was used for BPRPAM); and t is a time vector.

[0120] As expected, a gradual decrease in signal for all imaging agents was observed over time in E.G7-OVA tumor-mimicking phantoms, with the targeted signals showing higher intensity than the untargeted agent, IgG-IRD800 (FIG. 5(b)). Representative images from the final time point at 120 minutes are shown in FIG. 5(c). From these images, STP BP maps were generated to reveal the spatial distribution of PDL1, PD1, and CD80 concentrations within the E.G7-OVA tumor-mimicking phantom (FIG. 5(d)). Representative RPAM curves for PDL1, PD1, and CD80 are shown in FIG. 9.Single timepoint (STP) BP maps

[0121] STP BP maps for PDL1, PD1, and CD80 at the 120 minute-timepoint were created using the following equation:Equation 5:where STis the signal from the targeted image agent; Scis the signal from the untargeted imaging agent; and ttis any single imaging time point post rinsing.Example 9

[0122] To account for the dynamic expression of immune checkpoints in E.G7-OVA cells, QFC experiments - were run in parallel and compared to receptor numbers determined by mPAlR (FIG. 5(a)).

[0123] To validate the accuracy of mPAIR, a single population of E.G7-OVA cells was split into two groups: one for mPAIR experiments and the other for validation studies by QFC. This parallel approach allowed direct comparison of the number of PDL1, PD1, and CD80 receptors obtained from mPAIR to receptors measured by QFC. E.G7-OVA cells from the same flask were split into two groups. The first group was separated for the E.G7-OVA tumor-mimicking phantom experiments, and the second group was separated for quantitative flow cytometry (QFC) experiments to validate the number of receptors in the E.G7-OVA phantom determinedby mPAIR. QFC experiments, as described in Example 1, were run concurrently with the E.G7-OVA tumor-mimicking experiments (FIG. 5 panels (a) and (e)).

[0124] To assess agreement between observed receptor concentrations from mPAIR and expected values from QFC, Lin’s CCC (pc) was determined. The coefficient was calculated to be 0.75, which describes the deviation from the best-fit line (Pearson’s, r = 0.85) multiplied by the accuracy of the best-fit line compared to the line of identity (bias correction factor, Cb= 0.89). Thus, the Lin’s CCC indicates a strong, positive correlation between mPAIR and QFC. The overall RMSE for evaluating mPAIR against QFC is 0.48 log-receptors per population (FIG. 5(e)). Furthermore, the Bland-Atman plot illustrates that there is a systematic bias in the gold standard quantitative flow cytometry measurement (shown in FIG. 10).

[0125] In these experiments, the mPAIR method successfully quantified and assessed the spatial distribution of PDL1, PD1, and CD80 receptors in E.G7-OVA tumor-mimicking phantoms. Importantly, Lin’s CCC and its subcomponents (accuracy and precision), were high for all three receptors, indicating a strong positive and linear correlation between receptor numbers obtained from mPAIR and QFC (FIG. 5(e)).

[0126] The comparison, however, also revealed a slight discrepancy. The RMSE between receptor concentrations determined from each method was 0.48, suggesting minor deviation between observed versus expected receptor values. Several factors may account for this discrepancy. The Maestro imaging system used for mPAIR utilizes a charge-coupled device imaging sensor, which has a lower detection sensitivity than the avalanche photodiode detectors used by the Cytek Aurora system for QFC. The mPAIR experiments also used a high density of E.G7-OVA cells (n = 30 million) in each tumor-mimicking phantom, which could have posed physical limitations on the ability of each imaging agent to uniformly access every receptor. Additionally, these cells were also left to incubate with the imaging agents at room temperature to preserve the gel-like consistency of the agar, while cells used for QFC were prepared at 4°C to optimize conditions for flow cytometry and minimize cellular processes (i.e., receptor internalization).

[0127] While these factors likely contributed to the observed differences between mPAIR and QFC receptor numbers, the possibility of a bias with the QFC data was also investigated. The Bland-Altman plot shows that although most differences fall within the limits of agreement,there is a bias in the QFC data to systematically overestimate results, showing that even gold-standard methods are imperfect.Example 9

[0128] In this example, it was demonstrated that the present mPAIR method quantifies receptor concentrations of PDL1, PD1, and CD80 in an in vivo model using syngeneic murine tumor models in immunocompetent mice.

[0129] The E.G7OVA tumor model was selected for in vivo testing due to its detectable expression of the PDL1 axis proteins and successful model validation in phantom studies.

[0130] All studies using the syngeneic murine tumor models were approved by the Institutional Animal Care and Use Committee at Dartmouth College. Eight-week-old male and female C57BL / 6J mice were purchased from Charles River Laboratories (Wilmington, MA). For the first cohort of mice, 1 X 106E.G7OVA cells were implanted in the right flank of the mouse using 50 uL of serum free RPMI media. For the second and third cohorts of mice, 5 X 106E.G7OVA cells were implanted. Mice switched to a chlorophyll -free diet at least a week prior to imaging experiments. A summary of the experimental conditions used in all in vivo E.G7OVA trials is provided in the following Table. Male mice are designated as [1] and female mice are designated as [2], and LLP, RRP, etc. are identifiers for individual mice.

[0131] A 200 uL bolus of the mPAIR cocktail (three targeted fluorescent agents: IRD680-PD1, AF700-CD80, AF750-PDL1, and one non-targeted fluorescent agent: IRD800-IgG) was administered via tail-vein injection approximately 17 hours prior to mPAIR imaging, based on optimal timing reported by Sadeghipour et al., Prediction of optimal contrast times post-imaging agent administration to inform personalized fluorescence-guided surgery, J. Biomed. Opt. 25, (2020). Mice were anesthetized during imaging procedures. The skin covering the tumor and adjacent leg muscle regions were surgically excised prior to imaging.

[0132] An initial pilot study was conducted to test the signal outputs of the mPAIR composition when injected in mice ([1] LLP and [1] RRP). Based on these preliminary results, in the second cohort of mice, the concentration of CD80-AF700 was reduced in the mPAIR composition to improve the balance between signals ([1] NP; [1] LP; [1] RP). After removing the NIR filter embedded in the imaging system, the concentrations of all imaging agents were lowered to account for the improved signal transmission ([1] RRP; [2] RP; [2] LP). These iterative adjustments provided the mPAIR method with more accurate in vivo quantification of the PDL1 axis proteins.

[0133] For each mouse, the tumor and adjacent muscle regions were surgically exposed and imaged prior to processing the tumor for QFC (FIG. 11 panel (a)). Representative raw fluorescence emission spectra from a sample mouse are shown in FIG. 11 panel (b). The acquired spectral data were unmixed on a pixel-by-pixel basis using the multi-spectral unmixing algorithm described to isolate the individual contributions of each imaging agent.

[0134] Brightfield images of E.G7OVA tumor and muscle regions were obtained on an iPhone camera (Apple, Cupertino, CA). Multispectral images of the E.G7OVA tumors were collected using the Maestro M-MSI-500-FLEX imaging system, as described above. Autofluorescence images were collected from (n = 3) tumor-bearing mice not administered with the mPAIR cocktail to obtain the autofluorescence basis spectra used in the spectral unmixing algorithm for E.G7OVA in vivo tumor studies.

[0135] Spectral unmixing of acquired emission data using least squares fitting was performed as described above. For in vivo studies, background correction was applied by subtracting background from autofluorescence signal and incorporating a free constant term in the linear fit. This constant accounts for baseline system noise (i.e. read noise) and improved the accuracy of the spectral unmixing algorithm in in vivo studies.

[0136] Fluorescence signal from all fluorescent agents in the mPAIR cocktail was detected in the tumor region with lower signals in the muscle region (FIG. 12, panels d, h). Representative STP BP maps were then generated to visualize the spatial distribution of PDL1, PD1, and CD80 concentrations within an E.G7OVA tumor (FIG. 13, panel (c)).

[0137] STP BP maps and values for PDL1, PD1, and CD80 were created using the following equation:Equation 5a:where STis the signal of the targeted image agent from the tumor ROI; SUTis the signal of the untargeted image agent from the tumor ROI; SMTis the signal of the targeted image agent from the leg muscle ROI; SMUTis the signal of the untargeted image agent from the tumor ROI; and t. is the time of imaging.

[0138] To assess the spatial variation in receptor availability, tumors were segmented into inner and outer ROIs, and BPs for PDL1, PD1, and CD80 were calculated separately for each region (FIG. 14, panel (a)). Since BP reflects receptor concentration, it provides a more accurate representation of tumor target levels than single targeted fluorescence intensity. Pearson correlation analysis revealed strong agreement between inner and outer BP values for all receptors (PDL1: r = 0.97, p < 0.0001; CD80: r = 0.90, p < 0.01; PD1: r = 0.79, p < 0.05), indicating that receptor availability was relatively uniform in E.G7OVA tumors (FIG. 14, panels b-d). From an imaging perspective, these findings indicated effective penetration by the imaging agents and confirmed that a binding-site barrier was not a limiting factor for mPAIR imaging agents in vivo. From a biological perspective, the spatial uniformity of BPs suggests that the PDL1 axis proteins were available for binding homogenously across the tumor. This observation is consistent with other findings that the majority of PDL1, PD1, and CD80 signal was attributed to the bulk tumor cell population rather than the infiltrating immune cell populations, validating the uniform distribution of these proteins observed across the tumor. Tumor processing and flow cytometry

[0139] Following imaging, tumors were processed for flow cytometry to quantify their global PDL1, PD1, and CD80 expression, with a portion of each tumor saved as frozen tissue for potential use in additional studies.

[0140] E.G7OVA tumors were isolated and digested in 0.5 mg / mL type IV collagenase for 30 minutes at 37°C while spinning via a magnetic stir bar. Tumors were then mechanically disrupted by a GentleMACS Tissue Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Sample staining for QFC is described in section 5.3.4.

[0141] Cells were resuspended in FACS buffer (without murine Fc block) and 7-AAD dye was added to exclude dead cells from analysis for the first and second cohorts of mice. For the third cohort of mice, Live / Dead Fixable Blue Dead dye (Thermo Fischer) was used to assess viability. Flow cytometry data of stained samples were acquired using the Cytek Aurora and analyzed with FlowJo software.

[0142] Given the wide range and non-linear distribution of receptor expression, the Spearman correlation was calculated to determine the agreement between binding potential and receptor values from the quantitative flow cytometry for each tumor. The correlation was calculated to be 0.72 (p < 0.05) for the second cohort of mice imaged with the NIR filter, 0.89 (p < 0.01) for the third cohort of mice imaged without the NIR filter, and 0.69 (p < 0.001) for a pooled dataset which included all mice regardless of filter status or imaging agent concentrations (FIG. 15, panels a-c). Since many of the values were clustered towards the lower left of the correlation plot in FIG. 15 panel (c), the data was log-transformed to better visualize the distribution. This approach excluded any receptor values that were undetected by either mPAIR or QFC and resulted in a correlation of 0.76 (p < 0.01; FIG. 15, panel (d)). In all cases, a strong positive correlation was observed, supporting the robustness and accuracy of the mPAIR method across experimental conditions.IF image staining and analysis

[0143] To further improve the mPAIR model and investigate potential sources of discrepancy between mPAIR and flow cytometry, IF was performed on frozen tumor tissue sections and the results were compared to the median receptor levels previously quantified by flow cytometry. A small portion of the tumors (~ 0.2 grams) from the mPAIR mice cohort was saved in optimum cutting temperature compound (Tissue-Tek, Torrance, CA) and immediately stored in the -80°C freezer. Tissue was prepared at approximately 10-pm frozen sections and fixed in -20°C methanol for 10 minutes. Slides were then washed three times with PBS and blocked with 3% PBS-BSA for 30 minutes. Slides were then incubated with either PDL1-AF488, PD1-AF488, and CD80-AF488 (1 :200) antibodies in the blocking solution at room temperature for 60minutes. Slides were washed five times in PBS and incubated with Goat Anti-Rabbit 546 secondary antibody (1 :200; Thermo Fisher) in blocking solution at room temperature for 60 minutes. Slides were then washed five times with PBS and incubated with a nuclear stain, Hoechst (1 :2000; Thermo Fisher). Slides were washed five times before being cover slipped with ProLong Diamond (Thermo Fischer).

[0144] Immunofluorescence (IF) images of tissue sections were acquired using a KEYENCE BZ-810 fluorescence microscope (KEYENCE Corporation, Osaka, Japan). Images were exported and analyzed using FIJI.

[0145] IF images were analyzed using FIJI. Individual fluorescence channels were first separated to isolate the individual fluorescence channels. For each image, the raw integrated density was measured. To normalize for cell number, signal from the Alexa Fluor 564 (AF564) channel was normalized by dividing it by the corresponding DAPI signal. Final corrected fluorescence intensity values were obtained by subtracting the normalized signal of the negative control (DAPI and AF564 stained only).Example 11

[0146] FIG. 16 shows (a) immunofluorescence (IF) images of tumor tissue sections taken at 20x magnification, with two representative fields shown for both inner and outer tumor regions

[0147] Hoechst (blue) was stained on every tissue. Each row corresponds to a distinct region (inner or outer), and each column represents the following staining conditions from left to right: Hoechst / secondary antibody only (red; negative control); Hoechst / PDLl primary antibody / secondary antibody; Hoechst / PDl primary antibody / secondary antibody; and Hoechst / CD80 primary antibody / secondary antibody.

[0148] Average IF signal intensities across the inner and outer tumor regions were 0.04 ± 0.01 for PDL1, 0.26 ± 0.01 for PD1, and nearly undetectable at 0.01 ± 0.01 for CD80 (FIG. 16, panels a, b). Therefore, IF revealed similar relative trends between fluorescence signals and flow-derived receptor levels, providing additional validation of the mPAIR method (FIG. 16, panels b, c).

[0149] The absence of CD80 detection in flow cytometry from some of the E.G7OVA tumors in the mPAIR cohort was noted, despite consistently detecting measurable levels of CD80 in tumor phantom model. It was hypothesized that residual mPAIR imaging agents might remainbound to receptors within the tumors even after tissue dissociation and processing, potentially interfering with subsequent antibody staining. To test this, flow cytometry data was reanalyzed from cohort 3 by spectrally unmixing flow data to assess the presence of residual IRD680, AF700, AF750, and IRD800 signals. Notably, signal intensity from all dyes was higher in the tumor samples than in the control, indicating that the mPAIR imaging agents were still present in cells (FIG. 17). Residual fluorescence signals from IRD680 and AF700 were notably higher across all mice compared to those from AF750 and IRD800, suggesting that PD1 and CD80 may have experienced greater blocking from bound imaging agents during flow cytometry relative to PDL1 and IgG (FIG. 17, panel (c)). These findings support the possibility that receptor occupancy by mPAIR imaging agents may have blocked antibody binding during flow cytometry staining.

[0150] To assess the impact of this potential interference, the QFC results were compared to the mPAIR signals for each corresponding receptor. AF750 signal was detected across all mice, yet receptor quantification by QFC only detected PDL1 in one mouse (FIG. 17, panel (b)). Both IRD680 and PD1 were detected across all mice, but at significantly lower levels than in mice not administered with the mPAIR agents (FIG. 17 panel (c), FIG. 18 panel (b)). Notably, the AF700 signal was consistently detected across all mice, despite the absence of CD80 receptors detected by flow for the third cohort of mice (FIG. 17, panel (d)). The receptor counts from all mPAIR mice were then compared to the average receptor values obtained from prior QFC experiments. Unpaired t-test showed significant differences in the detection of PDL1, PD1, and CD80 receptors between the pan immune and mPAIR E.G7OVA tumors. Specifically, pan immune mice exhibited an average of 60 ± 20 PDL1 receptors, 1.4 x 104± 0.4 x 104PD1 receptors, 370 ± 90 CD80 receptors per tumor population. In contrast, mPAIR-administered mice had significantly lower receptor counts, with 30 ± 50 PDL1 receptors, 2 X 103± 1 X 103PD1 receptors, and 30 ± 70 CD80 receptors (FIG. 18). These results support the hypothesis that residual imaging agents can block antibodies during re-staining, leading to an underestimation of receptor availability and introducing potential limitations when using flow cytometry or IF as a reference for mPAIR validation.

[0151] Despite reduced receptor detection by flow cytometry and IF, mPAIR still demonstrated strong correlation with expected receptor values. When comparing the distribution of BP valueswith log-transformed receptor counts from the QFC mice, we observed consistent trends: PD1 had the highest expression in E.G70VA tumors, followed by CD80 and then PDL1 (FIG. 19).Example 12mPAIR identifies colocalization of PDL1 with its binding partners in E.G7OVA tumors

[0152] In this example, the functional relationships within the PDL1 axis in the TME, the spatial colocalization of PDL1, PD1, and CD80 was examined in E.G7OVA tumors were explored. (FIG. 20). Previous reports have demonstrated that PD1 and PDL1 proximity can serve as a theragnostic biomarker for anti-PDl ICI treatment response in metastatic melanoma and advanced Merkel cell carcinoma.115 116While these studies evaluated PDL1 and PD1 proximity in tumor tissue sections, this analysis was extended to the whole TME in tumorbearing immunocompetent mice.

[0153] Across all mice, it was observed that PDL1 and PD1 colocalization exhibited the greatest variability in receptor spatial overlap (FIG. 21). This aligns with previous findings suggesting that PD1 :PDL1 interactions can vary significantly within the TME. In contrast, PDL1 and CD80 demonstrated consistently strong correlations (r > 0.70) across all mice, which is consistent with earlier findings where internalization assays revealed tightly coordinated trafficking of PDL1 and CD80 (FIG. 21, table a). While these co-traffi eking behaviors have not been previously reported, the present findings provide additional support for a close functional relationship between PDL1 and CD80 within the TME. Future microscopy studies will be conducted to confirm whether PDL1 and CD80 heterodimers are responsible for the observed colocalization patterns.

[0154] CD80 and PD1 showed predominantly weak or moderate correlations across all E.G7OVA tumors, which is consistent with the fact that these molecules are not direct binding partners and are often expressed on different immune cell types (FIG. 21).

[0155] FIGs. 22A-C show representative confocal images of soluble PD1 binding (22A), anti-PDL1 binding (22B), and colocalization of soluble PD1 and anti-PDLl antibody (22C) on melanoma tumors. FIGs. 23A-F demonstrate that PAFI quantified unbound, functional PDL1 receptors in lymphoma and macrophage-like tumor cells using soluble PD1. FIG. 23 A shows representative images of untargeted agent signals, and FIG. 23B shows targeted agent signals in lymphoma-mimicking phantoms with no cells, lymphoma cells (EG7.OVA), and lymphomacells (EG7.0VA) stimulated with interferon gamma to increase PDL1 expression. FIGs 23C and 23D are representative time-dependent fluorescent signals of targeted imaging agent and untargeted control in EG7.0VA cells with no stimulation (23C) or stimulation with interferon gamma (23D). FIG. 23E is a table listing PDL1, PD1, and CD80 receptors quantified using quantitative flow cytometry (QFC) of lymphoma (EG7.0VA) or macrophage-like tumor cells (RAW) and binding potential determined by PAFI. FIG. 23F shows a linear correlation of the number of PDL1 receptors in all trials determined by PAFI (in tumor phantoms) and QFC run in parallel. Pearson correlation (r) was calculated between the two methods.

[0156] Overall, these findings provide evidence for in vivo colocalization between PDL1 and its binding partners, PD1 and CD80

[0157] The present disclosure describes development of mPAIR in a syngeneic tumor model in immunocompetent mice. Binding potentials for PDL1, PD1, and CD80 quantified by mPAIR showed strong correlation with expected receptor values determined by QFC, even under conditions where residual agent binding likely interfered with post-imaging flow cytometry and IF. In addition, PDL1 exhibited consistently strong spatial correlation with CD80, and variable correlation to PD1. Overall, these findings demonstrate that mPAIR is a promising, multiplexed technique for real-time, in vivo quantification of the PDL1 axis proteins for future clinical applications.EXEMPLARY EMBODIMENTSMethods of Detecting Available PD-L1 And / Or Functional PD-L1 by Paired Agent Imaging

[0158] Embodiment Al . A method of detecting or quantifying available Programmed Death-Ligand 1 and / or functional Programmed Death-Ligand 1 in tissue of a sample or a subject, the method comprising: contacting the tissue with a targeted fluorescent agent that binds to available Programmed Death-Ligand 1 (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll) and does not specifically bind to PD-L1 complexed with PD-1 or CD80; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent; acquiring targeted fluorescence emission data for one or more emission wavelengths associated with the targeted fluorescent agent; and detecting orquantifying a-PD-Ll and / or f-PD-Ll in the tissue based on the targeted fluorescence emission data.[01591 Embodiment A2. The method of embodiment Al, further comprising: contacting the tissue with a non-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent; acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent; correcting the targeted fluorescence emission data using the non-targeted fluorescence data; and detecting or quantifying a-PD-Ll and / or f-PD-Ll in the tissue based on the corrected targeted fluorescence emission data.

[0160] Embodiment A3. The method of embodiment Al , wherein the targeted fluorescent agent comprises a binding agent and a fluorophore.

[0161] Embodiment A4. The method of embodiment A3, wherein the binding agent is selected from the group consisting of soluble proteins (such as soluble PD-1), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0162] Embodiment A5. The method of embodiment Al, further comprising displaying a visual image of the tissue comprising a spatial representation of a-PD-Ll and / or f-PD-Ll present in the tissue.

[0163] Embodiment A6. The method of embodiment A5, further comprising obtaining a white-light image of the tissue, and superimposing the visual image on the white-light image.

[0164] Embodiment A7. The method of embodiment Al, further comprising quantifying cell-associated concentration of a-PD-Ll and / or f-PD-Ll in the tissue.

[0165] Embodiment A8. The method of embodiment Al , further comprising generating an a-PD-Ll binding potential map and / or an f-PD-Ll binding potential map of the sample.

[0166] Embodiment A9. The method of embodiment Al or A2, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired by channels of a multichannel photodetector.

[0167] Embodiment A10. The method of embodiment Al or A2, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquiredtogether, and the method further comprises spectral unmixing of the targeted and non-targeted fluorescence emission data.[01681 Embodiment Al 1. The method of any of embodiments Al to A10, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

[0169] Embodiment A12. The method of embodiments All, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by mPAIR.

[0170] Embodiment A13. The method of any of embodiments Al to A 10, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises: contacting the tissue with a second targeted fluorescent agent, acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent; acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; and correcting the second targeted fluorescence emission data using the non-targeted fluorescence data.

[0171] Embodiment A14. The method of embodiment A13, wherein the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

[0172] Embodiment Al 5. The method of any of embodiments Al to A14, further comprising: contacting the tissue with a PD-l-targeted fluorescent agent and a CD80-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents; acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the PD-1- and CD80-targeted fluorescent agents; correcting the PD-l-emission data and CD80-emission data using the nontargeted fluorescence data; and detecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

[0173] Embodiment Al 6. The method of any of embodiments All to Al 5, further comprising providing a spatial and / or temporal image of a-PD-Ll and / or f-PD-Ll in the tissue.

[0174] Embodiment A 17. The method of embodiment A16, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

[0175] Embodiment Al 8. The method of any of embodiments Al 1 to Al 7, further comprising calculating a PD-L1 binding potential of the tissue using a paired-agent model, such as a rinsing paired-agent kinetic model (RPAM).

[0176] Embodiment Al 9. The method of embodiment Al 8, further comprising calculating a PD1 binding potential and or a CD80 binding potential using the RPAM.

[0177] Embodiment A20. The method of any of embodiments Al 3 to Al 9, wherein each of the targeted fluorescent agents comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0178] Embodiment A21. The method of any of embodiments Al to A20, wherein the tissue is a sample extracted from a subject.

[0179] Embodiment A22. The method of embodiment A21, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0180] Embodiment A23. The method of embodiment A21, wherein the sample is a fresh frozen sample.

[0181] Embodiment A24. The method of any of embodiments Al to A23, wherein the tissue is present in a living subject.

[0182] Embodiment A25. The method of embodiment A24, further comprising administering the targeted fluorescent agent and the non-targeted fluorescent agent to the subject.

[0183] Embodiment A26. The method of embodiment A25, wherein the targeted fluorescent agent and the non-targeted fluorescent agent are administered to the subject for a surgical procedure on the tissue.

[0184] Embodiment A27. The method of any of embodiments Al to A26, wherein a-PD-Ll and / or f-PD-Ll is dynamically assessed in real-time.

[0185] Embodiment A28. The method of any of embodiments Al to A27, wherein the nontargeted fluorescent agent comprises a non-specific agent selected from the group consisting ofsoluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0186] Embodiment A29. The method of any of embodiments Al to A28, wherein the nontargeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

[0187] Embodiment A30. The method of any of embodiments Al to A29, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.Methods of Predicting or Monitoring Response To Immunotherapy

[0188] Embodiment Bl. A method of predicting or monitoring response by a subject to immunotherapy, the method comprising: detecting or quantifying available Programmed Death-Ligand (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll) in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to a-PD-L1 and / or f-PD-Ll and does not specifically bind to PD-L1 complexed with PD-1 or CD80; and determining an a-PD-Ll score and / or a f-PD-Ll score for the tissue.

[0189] Embodiment B2. The method of embodiment Bl, further comprising determining a PD-L1 binding potential of the tissue, wherein the a-PD-Ll score and / or the f-PD-Ll score is determined from the PD-L1 binding potential.

[0190] Embodiment B3. The method of embodiment B 1 or B2, further comprising comparing the a-PD-Ll and / or f-PD-Ll score to an a-PD-Ll threshold value and / or a f-PD-Ll threshold value, and identifying the subject as a likely responder if the a-PD-Ll score and / or the f-PD-Ll score is greater than or equal to the a-PD-Ll threshold value and / or the f-PD-Ll threshold value, and / or identifying the subject as a likely non-responder if the a-PD-Ll score and / or the f-PD-Ll score is below the a-PD-Ll threshold value and / or the f-PD-Ll threshold value.

[0191] Embodiment B4. The method of embodiment B 1 or B2, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the a-PD-Ll score and / or the f-PD-Ll score.

[0192] Embodiment B5. The method of embodiment Bl or B2, wherein the tissue comprises tumor tissue.

[0193] Embodiment B6. The method of embodiment B 1 or B2, wherein the tissue comprises tumor-draining lymph node (TDLN) tissue.

[0194] Embodiment B7. The method of embodiment B 1 or B2, further comprising monitoring treatment of a patient undergoing a therapy that increases or decreases a-PD-Ll and / or f-PD-Ll.

[0195] Embodiment B8. The method of embodiment B 1 or B2, further comprising monitoring treatment of a patient undergoing anti-PD-1 therapy and / or anti-PD-Ll therapy and / or anti-CD80 therapy.

[0196] Embodiment B9. The method of embodiment B 1 or B2, further comprising administering an immunotherapy to the subject before and / or after detecting or quantifying a-PD-L1 and / or f-PD-Ll.

[0197] Embodiment B10. The method of embodiment B9, further comprising monitoring treatment of the subject with the immunotherapy.

[0198] Embodiment Bl 1. The method of embodiment B10, wherein the subject is identified as a responder if the a-PD-Ll score and / or the f-PD-Ll score is greater than or equal to an a-PD-L1 threshold value and / or a f-PD-Ll threshold value, and / or identifying the subject as a non-responder if the a-PD-Ll score and / or the f-PD-Ll score is below the a-PD-Ll threshold value and / or the f-PD-Ll threshold value.

[0199] Embodiment B 12. The method of embodiment Bl 1, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the a-PD-Ll score and / or the f-PD-Ll score.

[0200] Embodiment B 13. The method of any of embodiments Bl to Bl 2, further comprising: obtaining a sample comprising tissue from the subject; contacting the sample with a targeted fluorescent agent and a non-targeted fluorescent agent, wherein the targeted fluorescent agent binds to a-PD-Ll and / or f-PD-Ll and does not specifically bind to PD-L1 complexed with PD-1 or CD80; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent; acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with the targeted fluorescent agent; acquiring non-targeted fluorescence emissiondata for one or more emissions wavelengths associated with the non-targeted fluorescent agent; and correcting the targeted fluorescence emission data using the non-targeted fluorescence data.

[0201] Embodiment B 14. The method of embodiment B 13, wherein the sample comprises melanocytes.

[0202] Embodiment Bl 5. The method of embodiment B 13, wherein the sample comprises blood.

[0203] Embodiment Bl 6. The method of any of embodiments Bl to Bl 5, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

[0204] Embodiment Bl 7. The method of embodiment Bl 6, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by mPAIR.

[0205] Embodiment Bl 8. The method of any of embodiments Bl to B15, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises: contacting the tissue with a second targeted fluorescent agent; acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent; acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; and correcting the second targeted fluorescence emission data using the non-targeted fluorescence data.

[0206] Embodiment B 19. The method of embodiment B 18, wherein the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

[0207] Embodiment B20. The method of any of embodiments B13 to B l 9, comprising: contacting the tissue with a PD-l-targeted fluorescent agent and a CD80-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents; acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the a-PD-1- and CD80-targeted fluorescent agents; correcting the PD-l-emission data and CD80-emission data usingthe nontargeted fluorescence data; and detecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

[0208] Embodiment B21. The method of any of embodiments B 16 to B20, further comprising providing a spatial and / or temporal image of a-PD-Ll and / or f-PD-Ll in the tissue.

[0209] Embodiment B22. The method of embodiment B21, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

[0210] Embodiment B23. The method of any of embodiments B7 to B22, wherein the targeted fluorescent agent comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0211] Embodiment B24. The method of any of embodiment Bl to B23, wherein the subject has adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma, ependymoma, breast cancer, carcinoid tumor, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell and small cell, lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, kaposi’s sarcoma, melanoma, small intestine cancer, soft tissue sarcoma,stomach cancer, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, or Wilms’ tumor.

[0212] Embodiment B25. The method of any of embodiment Bl to B23, wherein the subject has melanoma.

[0213] Embodiment B26. The method of any of embodiment Bl to B23, wherein the subject has T-cell lymphoma.

[0214] Embodiment B27. The method of any of embodiment B13 to B26, wherein the non-targeted fluorescent agent comprises a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as aflfibodies and nanobodies), and mixtures thereof.

[0215] Embodiment B28. The method of any of embodiment B13 to B27, wherein the nontargeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

[0216] Embodiment B29. The method of any of embodiment B13 to B28, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.Methods of Analyzing Samples with Multiple Fluorescent Agents & Spectral Unmixing

[0217] Embodiment Cl. A method of detecting or quantifying a panel of biomarkers in tissue of a sample or subject, comprising: contacting the tissue with a set of targeted fluorescent agents that bind to the biomarkers and a non-targeted fluorescent agent, wherein each of the targeted fluorescent agents and the non-target fluorescent agent has a different emission spectrum; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by each of the targeted fluorescent agents and the non-targeted fluorescent agent; acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with each of the targeted fluorescent agents; acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent; performing spectral unmixing of the targeted fluorescence emission data and non-targeted fluorescence emission data; correcting the targeted fluorescence emission data based on thenon-targeted fluorescence data; and detecting or quantifying each of the biomarkers of the panel in the tissue based on the spectrally-unmixed corrected targeted fluorescence emission data.

[0218] Embodiment C2. The method of embodiment Cl, wherein the set of targeted fluorescent agents comprises 3, 4, 5, 6 or more targeted fluorescent agents.

[0219] Embodiment C3. The method of embodiment Cl or C2, further comprising obtaining or determining reference fluorescence emission data for each of the targeted fluorescent agents and for the non-targeted fluorescent agent.

[0220] Embodiment C4. The method of embodiment C3, wherein the reference fluorescence emission data are determined for each of the targeted fluorescent agents and for the non-targeted fluorescent agent in a biologically relevant solvent, such as a lipid emulsion.

[0221] Embodiment C5. The method of embodiment C3, wherein the reference fluorescence emission data is used for the spectral unmixing.

[0222] Embodiment C6. The method of any of embodiments Cl to C5, wherein the fluorescence emission data is acquired as multispectral data.

[0223] Embodiment C7. The method of any of embodiments A2, A13, A15, B13, B15, B20, and Cl to C6, wherein the targeted fluorescence emission data is corrected by subtracting and normalizing by the non-targeted fluorescence data.

[0224] Embodiment C8. The method of any of embodiments A2, A13, A15, B13, B15, B20, and Cl to C6, wherein the targeted fluorescence emission data is corrected using a ratiometric calculation.

[0225] Embodiment C9. The method of embodiment C8, where the ratiometric calculation comprises dividing the targeted fluorescence emission at one or more time points by an intensity corrected untargeted fluorescence emission at the same one or more time point minus one.

[0226] Embodiment CIO. The method of embodiment C9, wherein the intensity corrected untargeted fluorescence emission is determined by multiplying the untargeted fluorescence emission by a ratio of targeted fluorescence emission to untargeted fluorescence emission collected from (a) a standard without the biomarker of interest at the same time point, or (b) from an earlier time point where concentrations of the targeted fluorescent agents and the nontargeted fluorescent agents are essentially the same.

[0227] Embodiment Cll. The method of any of embodiments A2, A13, A15, B13, B15, B20, and Cl to C6, wherein the targeted fluorescence emission data is corrected by fittingsequences of normalized targeted and untargeted fluorescence emission intensities with a nonlinear least-squares algorithm.[02281 Embodiment C12. The method of any of embodiments Cl to Cll, wherein the spectral unmixing comprises representing the targeted fluorescence emission data and the nontargeted fluorescence emission data as a linear combination of each of the fluorescent agents.

[0229] Embodiment C13. The method of embodiment Cl 2, further comprising performing a least-squares fitting to calculate relative contributions of each of the fluorescent agents.

[0230] Embodiment C14. The method of embodiment C13, wherein the step of acquiring fluorescence emission data comprises acquiring a set of multispectral images of the sample, wherein each of the multispectral images corresponds to a different wavelength band or range.

[0231] Embodiment C15. The method of embodiment C13 or C14, further comprising generating a three-dimensional (m x n x 1) image cube from each acquisition of fluorescence emission data, where m is the number of row-pixels, n is the number of column-pixels, and 1 is the number of wavelengths.

[0232] Embodiment C16. The method of embodiment Cl 5, wherein a measured signal intensity at wavelength for each of the fluorescent agents at each pixel of each of the multispectral images is determined by the formula:where x is the relevant amount of the fluorescent agent z, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent, where x is the relevant amount of the fluorescent agent i, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent.

[0233] Embodiment C17. The method of any of embodiments Cl to Cl 6, wherein the sample is illuminated with a broadband lamp filtered through a multiband excitation filter at stimulus wavelength(s) in bands or ranges of from about 535 to about 545 nm, from about 620 to about 645 nm, and from about 740 to about 750 nm.

[0234] Embodiment Cl 8. The method of any of embodiments Cl to Cl 6, wherein the multiband excitation filter comprises passbands of from about 538 to about 543 nm, from about 623 to about 641 nm, and from about 743 to about 747 nm.

[0235] Embodiment Cl 9. The method of any of embodiments Cl to Cl 8, wherein the fluorescence emission data is acquired through a multiband emission filter to obtain narrowband images in 5 nm increments from about 560 to about 860 nm.

[0236] Embodiment C20. The method of any of embodiments Cl to Cl 9, wherein the fluorescence emission data is acquired through a liquid crystal tunable filter.

[0237] Embodiment C21. The method of any of embodiments Cl to C20, wherein the fluorescence emission data is acquired in bands or ranges of from about 555 to about 610 nm, from about 655 to about 730 nm, and from about 760 to about 850 nm.

[0238] Embodiment C22. The method of any of embodiments Cl to C20, wherein the fluorescence emission data is acquired in bands or ranges of from about 557 to about 602, from about 660 to about 722, and from about 768 to about 846 nm.

[0239] Embodiment C23. The method of any of embodiments Cl to C22, wherein each of the targeted fluorescent agents comprises a fluorophore selected from the group consisting of optical dyes, such as OregonFluor 550 (OF550), OregonFluor 650 (OF650), IRDye 680LT (IR680), Alexa Fluor 700 (AF700), Alexa Fluor 750 (AF750), and IRDye 800CW (IR800).

[0240] Embodiment C24. The method of any of embodiments Cl to C22, wherein at least one of the targeted fluorescent agents comprises OregonFluor 550 (OF550).

[0241] Embodiment C25. The method of any of embodiments any of embodiments Cl to C22, wherein at least one of the targeted fluorescent agents comprises OregonFluor 650 (OF650).

[0242] Embodiment C26. The method of any of embodiments any of embodiments Cl to C22, wherein at least one of the targeted fluorescent agents comprises IRDye 680LT (IR680).

[0243] Embodiment C27. The method of any of embodiments any of embodiments Cl to C22, wherein at least one of the targeted fluorescent agents comprises Alexa Fluor 700 (AF700).

[0244] Embodiment C28. The method of any of embodiments any of embodiments Cl to C22, wherein at least one of the targeted fluorescent agents comprises Alexa Fluor 750 (AF750).

[0245] Embodiment C29. The method of any of embodiments any of embodiments Cl to C22, wherein an untargeted fluorescent agents comprises IRDye 800CW (IR800).

[0246] Embodiment C30. The method of any of embodiments Cl to C29, further comprising acquiring autofluorescence emission data prior to contacting the sample with the set of targeted fluorescent agents and the non-targeted fluorescent agent, and correcting the targeted fluorescence emission data using the autofluorescence emission data.Methods of Detecting PD-L1 Axis Proteins by Paired Agent Imaging

[0247] Embodiment DI . A method of detecting or quantifying a Programmed Death-Ligand 1 axis protein in tissue of a sample or a subject, the method comprising: contacting the tissue with a targeted fluorescent agent that binds to a Programmed Death-Ligand 1 (PD-L1) axis protein; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent; acquiring targeted fluorescence emission data for one or more emission wavelengths associated with the first targeted fluorescent agent; and detecting or quantifying the PD-L1 axis protein in the tissue based on the targeted fluorescence emission data.

[0248] Embodiment D2. The method of embodiment DI, further comprising: contacting the tissue with a non-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent; acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent; correcting the first and second fluorescence emission data using the non-targeted fluorescence data; and detecting or quantifying the PD-L1 axis protein in the tissue based on the corrected targeted fluorescence emission data.

[0249] Embodiment D3. The method of embodiment DI or D2, wherein the PD-L1 axis protein is PD-L1.

[0250] Embodiment D4. The method of embodiment DI or D2, wherein the PD-L1 axis protein is PD-1.

[0251] Embodiment D5. The method of embodiment DI or D2, wherein the PD-L1 axis protein is CD80.

[0252] Embodiment D6. The method of any of embodiments DI to D5, wherein the targeted fluorescent agent comprises a binding agent and a fluorophore.

[0253] Embodiment D7. The method of embodiment D6, wherein the binding agent is selected from the group consisting of soluble proteins (such as soluble PD-1), antibodies,antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0254] Embodiment D8. The method of any of embodiments DI to D7, further comprising displaying a visual image of the tissue comprising a spatial representation of the PD-L1 axis protein present in the tissue.

[0255] Embodiment D9. The method of embodiment D8, further comprising obtaining a white-light image of the tissue, and superimposing the visual image on the white-light image.

[0256] Embodiment DIO. The method of any of embodiments DI to D9, further comprising quantifying cell-associated concentration of the PD-L1 axis protein.

[0257] Embodiment Dll. The method of any of embodiments DI to DIO, further comprising generating a PD-L1 binding potential map of the sample.

[0258] Embodiment D12. The method of any of embodiments D2 to DI 1, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired by channels of a multichannel photodetector.

[0259] Embodiment DI 3, The method of any of embodiments D2 to DI 1, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired together, and the method further comprises spectral unmixing of the targeted and nontargeted fluorescence emission data.

[0260] Embodiment D14. The method of any of embodiments DI to D13, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with the PD-L1 axis protein by multi -spectral paired-agent imaging (PAI) of receptors (mPAIR).

[0261] Embodiment D15. The method of embodiments D14, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with PD-L1 by mPAIR.

[0262] Embodiment DI 6. The method of any of embodiments D2 to DI 5, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises: contacting the tissue with a second targeted fluorescent agent; acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent; acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; andcorrecting the second targeted fluorescence emission data using the non-targeted fluorescence data.[02631 Embodiment DI 7. The method of embodiment DI 6, further comprising: contacting the tissue with a third targeted fluorescent agent; acquiring third fluorescence emission data for one or more emissions wavelengths associated with the third targeted fluorescent agent; illuminating the tissue with light of stimulus wavelength(s) absorbed by the third targeted fluorescent agent; acquiring third targeted fluorescence emission data for one or more emissions wavelengths associated with the third targeted fluorescent agent; and correcting the third targeted fluorescence emission data using the non-targeted fluorescence data.

[0264] Embodiment DI 8. The method of embodiment DI 7, wherein the first targeted fluorescent agent binds to PD-L1, and the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1), and the third targeted fluorescent agent binds to Cluster of Differentiation 80 (CD80).

[0265] Embodiment DI 9. The method of any of embodiments DI to DI 8, further comprising providing a spatial and / or temporal image of PD-L1 in the tissue.

[0266] Embodiment D20. The method of embodiment D19, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

[0267] Embodiment D21. The method of any of embodiments D2 to D20, further comprising calculating a PD-L1 binding potential of the tissue using a paired-agent model, such as a rinsing paired-agent kinetic model (RPAM).

[0268] Embodiment D22. The method of embodiment D21, further comprising calculating a PD1 binding potential and or a CD80 binding potential using the paired-agent model.

[0269] Embodiment D23. The method of any of embodiments DI to D22, wherein each of the targeted fluorescent agents comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0270] Embodiment D24. The method of any of embodiments DI to D23, wherein the tissue is a sample extracted from a subject.

[0271] Embodiment D25. The method of embodiment D24, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0272] Embodiment D26. The method of embodiment D25, wherein the sample is a fresh frozen sample.

[0273] Embodiment D27. The method of any of embodiments DI to D26, wherein the tissue is present in a living subject.

[0274] Embodiment D28. The method of embodiment D27, further comprising administering the targeted fluorescent agent and the non-targeted fluorescent agent to the subject.

[0275] Embodiment D29. The method of embodiment D28, wherein the targeted fluorescent agent and the non-targeted fluorescent agent are administered to the subject for a surgical procedure on the tissue.

[0276] Embodiment D30. The method of any of embodiments DI to D29, wherein PD-L1 is dynamically assessed in real-time.

[0277] Embodiment D31. The method of any of embodiments DI to D30, wherein the nontargeted fluorescent agent comprises a fluorophore only, or a fluorophore and a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

[0278] Embodiment D32. The method of any of embodiments DI to D31, wherein the nonspecific agent has a molecular weight within 10% of the molecular weight of the binding agent of the targeted fluorescent agent.

[0279] Embodiment D33. The method of any of embodiments DI to D32, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.Methods of Predicting or Monitoring Response To Immunotherapy

[0280] Embodiment El . A method of predicting or monitoring response by a subject to cancer immunotherapy, the method comprising: obtaining a sample comprising tissue from the subject; contacting the sample with a targeted fluorescent agent and a non-targeted fluorescent agent, wherein the targeted fluorescent agent binds to an immune checkpoint biomarker;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent; acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with the targeted fluorescent agent; acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent; correcting the targeted fluorescence emission data using the non-targeted fluorescence data; detecting or quantifying the immune checkpoint biomarker in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to the immune checkpoint biomarker; and determining an immune checkpoint biomarker score for the tissue.

[0281] Embodiment E2. The method of embodiment El, wherein the immune checkpoint biomarker is a Programmed Death-Ligand (PD-L1), Programmed Cell Death Protein 1 (PD1), Cluster of differentiation 80 (CD80), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), Cluster of differentiation 47 (CD47), Indoleamine 2,3 -dioxygenase 1 (IDO1), B7 Homolog 3 (B7-H3) or B7 Homolog 4 (B7-H4).

[0282] Embodiment E3. The method of embodiment El, wherein the immune checkpoint biomarker is a Programmed Death-Ligand (PD-L1) axis protein.

[0283] Embodiment E4. The method of embodiment E3, wherein the Programmed Death-Ligand (PD-L1) axis protein is PD-L1.

[0284] Embodiment E5. The method of embodiment El or E4, further comprising comparing the immune checkpoint biomarker score to an immune checkpoint biomarker threshold value, and identifying the subject as a likely responder if the immune checkpoint biomarker score is greater than or equal to the immune checkpoint biomarker threshold value, and / or identifying the subject as a likely non-responder if the immune checkpoint biomarker score is below the immune checkpoint biomarker threshold value.

[0285] Embodiment E6. The method of embodiment El or E4, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the immune checkpoint biomarker score.

[0286] Embodiment E7. The method of embodiment El or E4, wherein the tissue comprises tumor tissue.

[0287] Embodiment E8. The method of embodiment El or E4, wherein the tissue comprises tumor-draining lymph node (TDLN) tissue.

[0288] Embodiment E9. The method of embodiment El or E4, further comprising monitoring treatment of a patient undergoing a therapy that increases or decreases the immune checkpoint biomarker.

[0289] Embodiment E10. The method of embodiment El or E4, further comprising monitoring treatment of a patient undergoing immune checkpoint therapy.

[0290] Embodiment Ell. The method of embodiment El or E4, further comprising administering a cancer immunotherapy to the subject before and / or after detecting or quantifying the immune checkpoint biomarker.

[0291] Embodiment E12. The method of embodiment El 1, further comprising monitoring treatment of the subject with the cancer immunotherapy.

[0292] Embodiment E13. The method of embodiment E12, wherein the subject is identified as a responder if the immune checkpoint biomarker score is greater than or equal to an immune checkpoint biomarker threshold value, and / or identifying the subject as a non-responder if the immune checkpoint biomarker score is below the immune checkpoint biomarker threshold value.

[0293] Embodiment E14. The method of embodiment E12, further comprising maintaining or adjusting administration of the cancer immunotherapy to the subject based on the immune checkpoint biomarker score.

[0294] Embodiment E15. The method of any of embodiments El to E14, wherein the sample comprises melanocytes.

[0295] Embodiment E16. The method of any of embodiments E l to E14, wherein the sample comprises blood.

[0296] Embodiment E17. The method of any of embodiments El to E16, wherein the immune checkpoint biomarker is a first immune checkpoint biomarker, and the method comprises detecting or quantifying a second immune checkpoint biomarker in the tissue simultaneously with the first immune checkpoint biomarker by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

[0297] Embodiment El 8. The method of embodiment El 7, wherein the first immune checkpoint biomarker is PD-L1, and the second immune checkpoint biomarker is Programmed cell death protein 1 (PD-1) and / or Cluster of Differentiation 80 (CD80).

[0298] Embodiment E19. The method of any of embodiments El to E16, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises: contacting the tissue with a second targeted fluorescent agent; acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent; acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; and correcting the second targeted fluorescence emission data using the non-targeted fluorescence data.

[0299] Embodiment E20. The method of embodiment El 9, wherein the first targeted fluorescent agent binds to PD-L1, and the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

[0300] Embodiment E21. The method of any of embodiments El to E16, wherein the targeted fluorescent agent is a PD-L1 -targeted fluorescent agent, and the methods further comprises: contacting the tissue with a PD-l-targeted fluorescent agent and a CD80-targeted fluorescent agent; illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents; acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the a-PD-1- and CD80-targeted fluorescent agents; correcting the PD-l-emission data and CD80-emission data using the nontargeted fluorescence data; and detecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

[0301] Embodiment E22. The method of any of embodiments El to E21, further comprising providing a spatial and / or temporal image of the immune checkpoint biomarker in the tissue.

[0302] Embodiment E23. The method of embodiment E22, comprising providing a spatial and / or temporal image of PD-L1, PD1 and / or CD80 in the tissue.

[0303] Embodiment E24. The method of any of embodiments El to E23, comprising determining colocalization of two or more of the immune checkpoint biomarkers in the tissue.

[0304] Embodiment E25. The method of embodiment E24, wherein the method comprises determining: PD1 colocalization with PDL1; CD80 colocalization with PDL1; and / or PD1 colocalization with CD80.

[0305] Embodiment E26. The method of any of embodiment El to E25, wherein the subject has adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma, ependymoma, breast cancer, carcinoid tumor, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell and small cell, lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0306] Embodiment E27. The method of any of embodiment El to E25, wherein the subject has melanoma.

[0307] Embodiment E28. The method of any of embodiment El to E25, wherein the subject has T-cell lymphoma.

[0308] Embodiment E29. The method of any of embodiment El to E25, wherein each of the targeted fluorescent agent(s) and the non-targeted fluorescent agent comprises a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), singlechain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.[03091 Embodiment E30. The method of embodiment E29, wherein the non-targeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

[0310] Embodiment E31. The method of embodiment E29, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.

[0311] Embodiment Fl. The method of any of embodiments D2, D16, D17, and El to E31, wherein the targeted fluorescence emission data is corrected by subtracting and normalizing by the non-targeted fluorescence emission data.

[0312] Embodiment F2. The method of any of embodiments D2, D16, D17, and El to E31, wherein the targeted fluorescence emission data is corrected using a ratiometric calculation.

[0313] Embodiment F3. The method of embodiment F2, where the ratiometric calculation comprises dividing the targeted fluorescence emission at one or more time points by an intensity corrected untargeted fluorescence emission at the same one or more time point minus one.

[0314] Embodiment F4. The method of embodiment F3, wherein the intensity corrected untargeted fluorescence emission is determined by multiplying the untargeted fluorescence emission by a ratio of targeted fluorescence emission to untargeted fluorescence emission collected from (a) a standard without the biomarker of interest at the same time point, or (b) from an earlier time point where concentrations of the targeted fluorescent agents and the nontargeted fluorescent agents are essentially the same.

[0315] Embodiment F5. The method of any of embodiments D2, D16, D17, and El to E31, wherein the targeted fluorescence emission data is corrected by fitting sequences of normalized targeted and untargeted fluorescence emission intensities with a nonlinear leastsquares algorithm.

[0316] Embodiment F6. The method of any of embodiments Fl to F5, wherein the spectral unmixing comprises representing the targeted fluorescence emission data and the non-targeted fluorescence emission data as a linear combination of each of the fluorescent agents.

[0317] Embodiment F7. The method of embodiment F6, further comprising performing a least-squares fitting to calculate relative contributions of each of the fluorescent agents.

[0318] Embodiment F8. The method of embodiment F7, wherein the step of acquiring fluorescence emission data comprises acquiring a set of multispectral images of the sample, wherein each of the multispectral images corresponds to a different wavelength band or range.

[0319] Embodiment F9. The method of embodiment F7 or F8, further comprising generating a three-dimensional (m x n x 1) image cube from each acquisition of fluorescence emission data, where m is the number of row-pixels, n is the number of column-pixels, and 1 is the number of wavelengths.

[0320] Embodiment Fl 0. The method of embodiment F9, a measured signal intensity V) at wavelength for for each of the fluorescent agents at each pixel of each of the multispectral images is determined by the formula:where x is the relevant amount of the fluorescent agent z, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent.

[0321] Embodiment F 11. The method of any of embodiments Fl to F 10, wherein the sample is illuminated with a broadband lamp filtered through a multiband excitation filter at stimulus wavelength(s) in bands or ranges of from about 535 to about 545 nm, from about 620 to about 645 nm, and from about 740 to about 750 nm.

[0322] Embodiment Fl 2. The method of any of embodiments Fl to F10, wherein the multiband excitation filter comprises passbands of from about 538 to about 543 nm, from about 623 to about 641 nm, and from about 743 to about 747 nm.

[0323] Embodiment F 13. The method of any of embodiments Fl to Fl 2, wherein the fluorescence emission data is acquired through a multiband emission filter to obtain narrowband images in 5 nm increments from about 560 to about 860 nm.

[0324] Embodiment Fl 4. The method of any of embodiments Fl to F13, wherein the fluorescence emission data is acquired through a liquid crystal tunable filter.

[0325] Embodiment Fl 5. The method of any of embodiments Fl to F14, wherein the fluorescence emission data is acquired in bands or ranges of from about 555 to about 610 nm, from about 655 to about 730 nm, and from about 760 to about 850 nm.

[0326] Embodiment Fl 6. The method of any of embodiments Fl to Fl 4, wherein the fluorescence emission data is acquired in bands or ranges of from about 557 to about 602, from about 660 to about 722, and from about 768 to about 846 nm.

[0327] Embodiment Fl 7. The method of any of embodiments Fl to Fl 6, wherein each of the targeted fluorescent agents comprises a fluorophore selected from the group consisting of fluorescent dyes having emission wavelengths of about 550 nm, about 650 nm, about 680 nm, about 700 nm, about 750 nm, and about 800 nm.

[0328] Embodiment Fl 8. The method of any of embodiments Fl to Fl 6, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 550 nm.

[0329] Embodiment Fl 9. The method of any of embodiments any of embodiments Fl to Fl 6, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 650 nm.

[0330] Embodiment F20. The method of any of embodiments any of embodiments Fl to Fl 6, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 680 nm.

[0331] Embodiment F21. The method of any of embodiments any of embodiments Fl to Fl 6, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 700 nm.

[0332] Embodiment F22. The method of any of embodiments any of embodiments Fl to Fl 6, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 750 nm.

[0333] Embodiment F23. The method of any of embodiments any of embodiments Fl to Fl 6, wherein an untargeted fluorescent agents comprises a fluorophore having an emission wavelength of about 800 nm.

[0334] Embodiment F24. The method of any of embodiments Fl to F23, further comprising acquiring autofluorescence emission data prior to contacting the sample with the set of targeted fluorescent agents and the non-targeted fluorescent agent, and correcting the targeted fluorescence emission data using the autofluorescence emission data.

[0335] The present disclosure includes the text and figures set forth above and the claims which follow. All patents and publications referred to above expressly incorporated by reference. It isto be understood that the terminology used herein is for the purpose of describing various embodiments and is not intended to be limiting.

[0336] Technical and scientific terms used herein shall have the meaning(s) as commonly understood by those working in the fields to which this disclosure pertain. Defined terms are in addition to the technical and scientific meanings.

[0337] As used herein, the terms “a,” “an,” and “the” include both singular and plural referents, unless the context clearly dictates otherwise. Thus, for example, “a compound” includes one compound and plural compounds.

Claims

CLAIMSWe claim:

1. A method of detecting or quantifying available Programmed Death-Ligand 1 and / or functional Programmed Death-Ligand 1 in tissue of a sample or a subject, the method comprising:contacting the tissue with a targeted fluorescent agent that binds to available Programmed Death-Ligand 1 (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll) and does not specifically bind to PD-L1 complexed with PD-1 or CD80;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent;acquiring targeted fluorescence emission data for one or more emission wavelengths associated with the targeted fluorescent agent; anddetecting or quantifying a-PD-Ll and / or f-PD-Ll in the tissue based on the targeted fluorescence emission data.

2. The method of claim 1, further comprising:contacting the tissue with a non-targeted fluorescent agent;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent;acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent;correcting the targeted fluorescence emission data using the non-targeted fluorescence data; anddetecting or quantifying a-PD-Ll and / or f-PD-Ll in the tissue based on the corrected targeted fluorescence emission data.

3. The method of claim 1, wherein the targeted fluorescent agent comprises a binding agent and a fluorophore.

4. The method of claim 3, wherein the binding agent is selected from the group consisting of soluble proteins (such as soluble PD-1), antibodies, antibody fragments (such as Fv, Fab, Fab’,and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

5. The method of claim 1, further comprising displaying a visual image of the tissue comprising a spatial representation of a-PD-Ll and / or f-PD-Ll present in the tissue.

6. The method of claim 5, further comprising obtaining a white-light image of the tissue, and superimposing the visual image on the white-light image.

7. The method of claim 1, further comprising quantifying cell-associated concentration of a-PD-L1 and / or f-PD-Ll in the tissue.

8. The method of claim 1, further comprising generating an a-PD-Ll binding potential map and / or an f-PD-Ll binding potential map of the sample.

9. The method of claim 1 or 2, wherein the targeted fluorescence emission data and the nontargeted fluorescence emission data are acquired by channels of a multichannel photodetector.

10. The method of claim 1 or 2, wherein the targeted fluorescence emission data and the nontargeted fluorescence emission data are acquired together, andthe method further comprises spectral unmixing of the targeted and non-targeted fluorescence emission data.

11. The method of any of claims 1 to 10, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

12. The method of claims 11, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by mPAIR.

13. The method of any of claims 1 to 10, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises:contacting the tissue with a second targeted fluorescent agent;acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent;illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent;acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; andcorrecting the second targeted fluorescence emission data using the non-targeted fluorescence data.

14. The method of claim 13, wherein the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

15. The method of any of claims 1 to 14, further comprising:contacting the tissue with a PD-1 -targeted fluorescent agent and a CD80-targeted fluorescent agent;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents;acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the PD-1- and CD80-targeted fluorescent agents;correcting the PD-l-emission data and CD80-emission data using the non-targeted fluorescence data; anddetecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

16. The method of any of claims 11 to 15, further comprising providing a spatial and / or temporal image of a-PD-Ll and / or f-PD-Ll in the tissue.

17. The method of claim 16, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

18. The method of any of claims 11 to 17, further comprising calculating a PD-L1 binding potential of the tissue using a paired-agent model, such as a rinsing paired-agent kinetic model (RPAM).

19. The method of claim 18, further comprising calculating a PD1 binding potential and or a CD80 binding potential using the RPAM.

20. The method of any of claims 13 to 19, wherein each of the targeted fluorescent agents comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

21. The method of any of claims 1 to 20, wherein the tissue is a sample extracted from a subject.

22. The method of claim 21, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

23. The method of claim 21, wherein the sample is a fresh frozen sample.

24. The method of any of claims 1 to 23, wherein the tissue is present in a living subject.

25. The method of claim 24, further comprising administering the targeted fluorescent agent and the non-targeted fluorescent agent to the subject.

26. The method of claim 25, wherein the targeted fluorescent agent and the non-targeted fluorescent agent are administered to the subject for a surgical procedure on the tissue.

27. The method of any of claims 1 to 26, wherein a-PD-Ll and / or f-PD-Ll is dynamically assessed in real-time.

28. The method of any of claims 1 to 27, wherein the non-targeted fluorescent agent comprises a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

29. The method of any of claims 1 to 28, wherein the non-targeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

30. The method of any of claims 1 to 29, wherein the targeted fluorescent agent and the nontargeted fluorescent agent comprise antibodies of the same clonal isotype.

31. A method of predicting or monitoring response by a subject to immunotherapy, the method comprising:detecting or quantifying available Programmed Death-Ligand (a-PD-Ll) and / or functional Programmed Death-Ligand 1 (f-PD-Ll) in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to a-PD-Ll and / or f-PD-Ll and does not specifically bind to PD-L1 complexed with PD-1 or CD80; anddetermining an a-PD-Ll score and / or a f-PD-Ll score for the tissue.

32. The method of claim 31, further comprising determining a PD-L1 binding potential of the tissue, wherein the a-PD-Ll score and / or the f-PD-Ll score is determined from the PD-L1 binding potential.

33. The method of claim 31 or 32, further comprising comparing the a-PD-Ll and / or f-PD-Ll score to an a-PD-Ll threshold value and / or a f-PD-Ll threshold value, andidentifying the subject as a likely responder if the a-PD-Ll score and / or the f-PD-Ll score is greater than or equal to the a-PD-Ll threshold value and / or the f-PD-Ll threshold value, and / or identifying the subject as a likely non-responder if the a-PD-Ll score and / or the f-PD-Ll score is below the a-PD-Ll threshold value and / or the f-PD-Ll threshold value.

34. The method of claim 31 or 32, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the a-PD-Ll score and / or the f-PD-Ll score.

35. The method of claim 31 or 32, wherein the tissue comprises tumor tissue.

36. The method of claim 31 or 32, wherein the tissue comprises tumor-draining lymph node (TDLN) tissue.

37. The method of claim 31 or 32, further comprising monitoring treatment of a patient undergoing a therapy that increases or decreases a-PD-Ll and / or f-PD-Ll.

38. The method of claim 31 or 32, further comprising monitoring treatment of a patient undergoing anti-PD-1 therapy and / or anti-PD-Ll therapy and / or anti-CD80 therapy.

39. The method of claim 31 or 32, further comprising administering an immunotherapy to the subject before and / or after detecting or quantifying a-PD-Ll and / or f-PD-Ll .

40. The method of claim 39, further comprising monitoring treatment of the subject with the immunotherapy.

41. The method of claim 40, wherein the subject is identified as a responder if the a-PD-Ll score and / or the f-PD-Ll score is greater than or equal to an a-PD-Ll threshold value and / or a f-PD-L1 threshold value, and / or identifying the subject as a non-responder if the a-PD-Ll score and / or the f-PD-Ll score is below the a-PD-Ll threshold value and / or the f-PD-Ll threshold value.

42. The method of claim 41, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the a-PD-Ll score and / or the f-PD-Ll score.

43. The method of any of claims 31 to 42, further comprising:obtaining a sample comprising tissue from the subject;contacting the sample with a targeted fluorescent agent and a non-targeted fluorescent agent, wherein the targeted fluorescent agent binds to a-PD-Ll and / or f-PD-Ll and does not specifically bind to PD-L1 complexed with PD-1 or CD80;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent;acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with the targeted fluorescent agent;acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent; andcorrecting the targeted fluorescence emission data using the non-targeted fluorescence data.

44. The method of claim 43, wherein the sample comprises melanocytes.

45. The method of claim 43, wherein the sample comprises blood.

46. The method of any of claims 31 to 45, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

47. The method of claim 46, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with a-PD-Ll and / or f-PD-Ll by mPAIR.

48. The method of any of claims 31 to 45, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises:contacting the tissue with a second targeted fluorescent agent;acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent;illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent;acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; andcorrecting the second targeted fluorescence emission data using the non-targeted fluorescence data.

49. The method of claim 48, wherein the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

50. The method of any of claims 43 to 49, comprising:contacting the tissue with a PD-1 -targeted fluorescent agent and a CD80-targeted fluorescent agent;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents;acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the a-PD-1- and CD80-targeted fluorescent agents;correcting the PD-l-emission data and CD80-emission data using the non-targeted fluorescence data; anddetecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

51. The method of any of claims 46 to 50, further comprising providing a spatial and / or temporal image of a-PD-Ll and / or f-PD-Ll in the tissue.

52. The method of claim 51, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

53. The method of any of claims 37 to 52, wherein the targeted fluorescent agent comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-Ll, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

54. The method of any of claim 31 to 53, wherein the subject has adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma, ependymoma, breast cancer, carcinoid tumor, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell and small cell, lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, kaposi’s sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, or Wilms’ tumor.

55. The method of any of claim 31 to 53, wherein the subject has melanoma.

56. The method of any of claim 31 to 53, wherein the subject has T-cell lymphoma.

57. The method of any of claim 43 to 56, wherein the non-targeted fluorescent agent comprises a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

58. The method of any of claim 43 to 57, wherein the non-targeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

59. The method of any of claim 43 to 58, wherein the targeted fluorescent agent and the nontargeted fluorescent agent comprise antibodies of the same clonal isotype.

60. A method of detecting or quantifying a panel of biomarkers in tissue of a sample or subject, comprising:contacting the tissue with a set of targeted fluorescent agents that bind to the biomarkers and a non-targeted fluorescent agent, wherein each of the targeted fluorescent agents and the nontarget fluorescent agent has a different emission spectrum;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by each of the targeted fluorescent agents and the non-targeted fluorescent agent;acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with each of the targeted fluorescent agents;acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent;performing spectral unmixing of the targeted fluorescence emission data and non-targeted fluorescence emission data;correcting the targeted fluorescence emission data based on the non-targeted fluorescence data; anddetecting or quantifying each of the biomarkers of the panel in the tissue based on the spectrally-unmixed corrected targeted fluorescence emission data.

61. The method of claim 60, wherein the set of targeted fluorescent agents comprises 3, 4, 5, 6 or more targeted fluorescent agents.

62. The method of claim 60 or 61, further comprising obtaining or determining reference fluorescence emission data for each of the targeted fluorescent agents and for the non-targeted fluorescent agent.

63. The method of claim 62, wherein the reference fluorescence emission data are determined for each of the targeted fluorescent agents and for the non-targeted fluorescent agent in a biologically relevant solvent, such as a lipid emulsion.

64. The method of claim 62, wherein the reference fluorescence emission data is used for the spectral unmixing.

65. The method of any of claims 60 to 64, wherein the fluorescence emission data is acquired as multi spectral data.

66. The method of any of claims 2, 13, 15, 43, 45, 50, and 60 to 65, wherein the targeted fluorescence emission data is corrected by subtracting and normalizing by the non-targeted fluorescence data.

67. The method of any of claims 2, 13, 15, 43, 45, 50, and 60 to 65, wherein the targeted fluorescence emission data is corrected using a ratiometric calculation.

68. The method of claim 67, where the ratiometric calculation comprises dividing the targeted fluorescence emission at one or more time points by an intensity corrected untargeted fluorescence emission at the same one or more time point minus one.

69. The method of claim 68, wherein the intensity corrected untargeted fluorescence emission is determined by multiplying the untargeted fluorescence emission by a ratio of targeted fluorescence emission to untargeted fluorescence emission collected from (a) a standard withoutthe biomarker of interest at the same time point, or (b) from an earlier time point where concentrations of the targeted fluorescent agents and the non-targeted fluorescent agents are essentially the same.

70. The method of any of claims 2, 13, 15, 43, 45, 50, and 60 to 65, wherein the targeted fluorescence emission data is corrected by fitting sequences of normalized targeted and untargeted fluorescence emission intensities with a nonlinear least-squares algorithm.

71. The method of any of claims 60 to 70, wherein the spectral unmixing comprises representing the targeted fluorescence emission data and the non-targeted fluorescence emission data as a linear combination of each of the fluorescent agents.

72. The method of claim 71, further comprising performing a least-squares fitting to calculate relative contributions of each of the fluorescent agents.

73. The method of claim 72, wherein the step of acquiring fluorescence emission data comprises acquiring a set of multispectral images of the sample, wherein each of the multispectral images corresponds to a different wavelength band or range.

74. The method of claim 72 or 73, further comprising generating a three-dimensional (m x n x 1) image cube from each acquisition of fluorescence emission data, where m is the number of row-pixels, n is the number of column-pixels, and 1 is the number of wavelengths.

75. The method of claim 74, wherein a measured signal intensity (X) wavelength X for each of the fluorescent agents at each pixel of each of the multispectral images is determined by the formula:where x is the relevant amount of the fluorescent agent z, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent.

76. The method of any of claims 60 to 75, wherein the sample is illuminated with a broadband lamp filtered through a multiband excitation filter at stimulus wavelength(s) in bands or ranges of from about 535 to about 545 nm, from about 620 to about 645 nm, and from about 740 to about 750 nm.

77. The method of any of claims 60 to 75, wherein the multiband excitation filter comprises passbands of from about 538 to about 543 nm, from about 623 to about 641 nm, and from about 743 to about 747 nm.

78. The method of any of claims 60 to 77, wherein the fluorescence emission data is acquired through a multiband emission filter to obtain narrowband images in 5 nm increments from about 560 to about 860 nm.

79. The method of any of claims 60 to 78, wherein the fluorescence emission data is acquired through a liquid crystal tunable filter.

80. The method of any of claims 60 to 79, wherein the fluorescence emission data is acquired in bands or ranges of from about 555 to about 610 nm, from about 655 to about 730 nm, and from about 760 to about 850 nm.

81. The method of any of claims 60 to 79, wherein the fluorescence emission data is acquired in bands or ranges of from about 557 to about 602, from about 660 to about 722, and from about 768 to about 846 nm.

82. The method of any of claims 60 to 81, wherein each of the targeted fluorescent agents comprises a fluorophore selected from the group consisting of optical dyes, such as OregonFluor 550 (OF550), OregonFluor 650 (OF650), IRDye 680LT (IR680), Alexa Fluor 700 (AF700), Alexa Fluor 750 (AF750), and IRDye 800CW (IR800).

83. The method of any of claims 60 to 81, wherein at least one of the targeted fluorescent agents comprises OregonFluor 550 (OF550).

84. The method of any of claims any of claims 60 to 81, wherein at least one of the targeted fluorescent agents comprises OregonFluor 650 (OF650).

85. The method of any of claims any of claims 60 to 81, wherein at least one of the targeted fluorescent agents comprises IRDye 680LT (IR680).

86. The method of any of claims any of claims 60 to 81, wherein at least one of the targeted fluorescent agents comprises Alexa Fluor 700 (AF700).

87. The method of any of claims any of claims 60 to 81, wherein at least one of the targeted fluorescent agents comprises Alexa Fluor 750 (AF750).

88. The method of any of claims any of claims 60 to 81, wherein an untargeted fluorescent agents comprises IRDye 800CW (1R800).

89. The method of any of claims 60 to 88, further comprising acquiring autofluorescence emission data prior to contacting the sample with the set of targeted fluorescent agents and the non-targeted fluorescent agent, andcorrecting the targeted fluorescence emission data using the autofluorescence emission data.

90. A method of detecting or quantifying a Programmed Death-Ligand 1 axis protein in tissue of a sample or a subject, the method comprising:contacting the tissue with a targeted fluorescent agent that binds to a Programmed Death-Ligand 1 (PD-L1) axis protein;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent;acquiring targeted fluorescence emission data for one or more emission wavelengths associated with the first targeted fluorescent agent; anddetecting or quantifying the PD-L1 axis protein in the tissue based on the targeted fluorescence emission data.

91. The method of claim 90, further comprising:contacting the tissue with a non-targeted fluorescent agent;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent;acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent;correcting the first and second fluorescence emission data using the non-targeted fluorescence data; anddetecting or quantifying the PD-L1 axis protein in the tissue based on the corrected targeted fluorescence emission data.

92. The method of claim 90 or 91, wherein the PD-L1 axis protein is PD-L1.

93. The method of claim 90 or 91, wherein the PD-L1 axis protein is PD-1.

94. The method of claim 90 or 91, wherein the PD-L1 axis protein is CD80.

95. The method of any of claims 90 to 94, wherein the targeted fluorescent agent comprises a binding agent and a fluorophore.

96. The method of claim 95, wherein the binding agent is selected from the group consisting of soluble proteins (such as soluble PD-1), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

97. The method of any of claims 90 to 96, further comprising displaying a visual image of the tissue comprising a spatial representation of the PD-L1 axis protein present in the tissue.

98. The method of claim 97, further comprising obtaining a white-light image of the tissue, and superimposing the visual image on the white-light image.

99. The method of any of claims 90 to 98, further comprising quantifying cell-associated concentration of the PD-L1 axis protein.

100. The method of any of claims 90 to 99, further comprising generating a PD-L1 binding potential map of the sample.

101. The method of any of claims 91 to 100, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired by channels of a multichannel photodetector.

102. The method of any of claims 91 to 100, wherein the targeted fluorescence emission data and the non-targeted fluorescence emission data are acquired together, andthe method further comprises spectral unmixing of the targeted and non-targeted fluorescence emission data.

103. The method of any of claims 90 to 102, wherein the method comprises detecting or quantifying a receptor in the tissue simultaneously with the PD-L1 axis protein by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

104. The method of claims 103, wherein the method comprises detecting or quantifying PD-1 or CD80 in the tissue simultaneously with PD-L1 by mPAIR.

105. The method of any of claims 91 to 104, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises:contacting the tissue with a second targeted fluorescent agent,acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent;illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent;acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; andcorrecting the second targeted fluorescence emission data using the non-targeted fluorescence data.

106. The method of claim 105, further comprising:contacting the tissue with a third targeted fluorescent agent,acquiring third fluorescence emission data for one or more emissions wavelengths associated with the third targeted fluorescent agent;illuminating the tissue with light of stimulus wavelength(s) absorbed by the third targeted fluorescent agent;acquiring third targeted fluorescence emission data for one or more emissions wavelengths associated with the third targeted fluorescent agent; andcorrecting the third targeted fluorescence emission data using the non-targeted fluorescence data.

107. The method of claim 106, wherein the first targeted fluorescent agent binds to PD-L1, and the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1), and the third targeted fluorescent agent binds to Cluster of Differentiation 80 (CD80).

108. The method of any of claims 90 to 107, further comprising providing a spatial and / or temporal image of PD-L1 in the tissue.

109. The method of claim 108, further comprising providing a spatial and / or temporal image of PD1 and / or CD80 in the tissue.

110. The method of any of claims 91 to 109, further comprising calculating a PD-L1 binding potential of the tissue using a paired-agent model, such as a rinsing paired-agent kinetic model (RPAM).

111. The method of claim 110, further comprising calculating a PD1 binding potential and or a CD80 binding potential using the paired-agent model.

112. The method of any of claims 90 to 111, wherein each of the targeted fluorescent agents comprises a binding agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

113. The method of any of claims 90 to 112, wherein the tissue is a sample extracted from a subject.

114. The method of claim 113, wherein the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

115. The method of claim 114, wherein the sample is a fresh frozen sample.

116. The method of any of claims 90 to 115, wherein the tissue is present in a living subject.

117. The method of claim 116, further comprising administering the targeted fluorescent agent and the non-targeted fluorescent agent to the subject.

118. The method of claim 117, wherein the targeted fluorescent agent and the non-targeted fluorescent agent are administered to the subject for a surgical procedure on the tissue.

119. The method of any of claims 90 to 118, wherein PD-L1 is dynamically assessed in realtime.

120. The method of any of claims 90 to 119, wherein the non-targeted fluorescent agent comprises a fluorophore only, or a fluorophore and a non-specific agent selected from the groupconsisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

121. The method of any of claims 90 to 120, wherein the non-specific agent has a molecular weight within 10% of the molecular weight of the binding agent of the targeted fluorescent agent.

122. The method of any of claims 90 to 121, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.

123. A method of predicting or monitoring response by a subject to cancer immunotherapy, the method comprising:obtaining a sample comprising tissue from the subject;contacting the sample with a targeted fluorescent agent and a non-targeted fluorescent agent, wherein the targeted fluorescent agent binds to an immune checkpoint biomarker;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the targeted fluorescent agent and the non-targeted fluorescent agent;acquiring targeted fluorescence emission data for one or more emissions wavelengths associated with the targeted fluorescent agent;acquiring non-targeted fluorescence emission data for one or more emissions wavelengths associated with the non-targeted fluorescent agent;correcting the targeted fluorescence emission data using the non-targeted fluorescence data;detecting or quantifying the immune checkpoint biomarker in tissue of the subject based on fluorescence emission data from a targeted fluorescent agent that binds to the immune checkpoint biomarker; anddetermining an immune checkpoint biomarker score for the tissue.

124. The method of claim 123, wherein the immune checkpoint biomarker is a Programmed Death-Ligand (PD-L1), Programmed Cell Death Protein 1 (PD1), Cluster of differentiation 80(CD80), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), B and T lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), Cluster of differentiation 47 (CD47), Indoleamine 2,3-dioxygenase 1 (IDO1), B7 Homolog 3 (B7-H3) or B7 Homolog 4 (B7-H4).

125. The method of claim 123, wherein the immune checkpoint biomarker is a Programmed Death-Ligand (PD-L1) axis protein.

126. The method of claim 125, wherein the Programmed Death-Ligand (PD-L1) axis protein is PD-Ll.

127. The method of claim 123 or 126, further comprising comparing the immune checkpoint biomarker score to an immune checkpoint biomarker threshold value, andidentifying the subject as a likely responder if the immune checkpoint biomarker score is greater than or equal to the immune checkpoint biomarker threshold value, and / or identifying the subject as a likely non-responder if the immune checkpoint biomarker score is below the immune checkpoint biomarker threshold value.

128. The method of claim 123 or 126, further comprising maintaining or adjusting administration of the immunotherapy to the subject based on the immune checkpoint biomarker score.

129. The method of claim 123 or 126, wherein the tissue comprises tumor tissue.

130. The method of claim 123 or 126, wherein the tissue comprises tumor-draining lymph node (TDLN) tissue.

131. The method of claim 123 or 126, further comprising monitoring treatment of a patient undergoing a therapy that increases or decreases the immune checkpoint biomarker.

132. The method of claim 123 or 126, further comprising monitoring treatment of a patient undergoing immune checkpoint therapy.

133. The method of claim 123 or 126, further comprising administering a cancer immunotherapy to the subject before and / or after detecting or quantifying the immune checkpoint biomarker.

134. The method of claim 133, further comprising monitoring treatment of the subject with the cancer immunotherapy.

135. The method of claim 134, wherein the subject is identified as a responder if the immune checkpoint biomarker score is greater than or equal to an immune checkpoint biomarker threshold value, and / or identifying the subject as a non-responder if the immune checkpoint biomarker score is below the immune checkpoint biomarker threshold value.

136. The method of claim 134, further comprising maintaining or adjusting administration of the cancer immunotherapy to the subject based on the immune checkpoint biomarker score.

137. The method of any of claims 123 to 136, wherein the sample comprises melanocytes.

138. The method of any of claims 123 to 136, wherein the sample comprises blood.

139. The method of any of claims 123 to 138, wherein the immune checkpoint biomarker is a first immune checkpoint biomarker, and the method comprises detecting or quantifying a second immune checkpoint biomarker in the tissue simultaneously with the first immune checkpoint biomarker by multi-spectral paired-agent imaging (PAI) of receptors (mPAIR).

140. The method of claim 139, wherein the first immune checkpoint biomarker is PD-L1, and the second immune checkpoint biomarker is Programmed cell death protein 1 (PD-1) and / or Cluster of Differentiation 80 (CD80).

141. The method of any of claims 123 to 138, wherein the targeted fluorescent agent is a first targeted fluorescent agent, and the method further comprises:contacting the tissue with a second targeted fluorescent agent,acquiring second fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent;illuminating the tissue with light of stimulus wavelength(s) absorbed by the second targeted fluorescent agent and the non-targeted fluorescent agent;acquiring second targeted fluorescence emission data for one or more emissions wavelengths associated with the second targeted fluorescent agent; andcorrecting the second targeted fluorescence emission data using the non-targeted fluorescence data.

142. The method of claim 141, wherein the first targeted fluorescent agent binds to PD-L1, and the second targeted fluorescent agent binds to Programmed cell death protein 1 (PD-1) or to Cluster of Differentiation 80 (CD80).

143. The method of any of claims 123 to 138, wherein the targeted fluorescent agent is a PD-L1 -targeted fluorescent agent, and the methods further comprises:contacting the tissue with a PD-1 -targeted fluorescent agent and a CD80-targeted fluorescent agent;illuminating the tissue one or more times with light of stimulus wavelength(s) absorbed by the PD-1- and CD80-targeted fluorescent agents;acquiring PD-l-emission data and CD80-emission data for one or more emissions wavelengths associated with the a-PD-1- and CD80-targeted fluorescent agents;correcting the PD-l-emission data and CD80-emission data using the non-targeted fluorescence data; anddetecting or quantifying PD-1 and CD80 in the tissue based on the corrected PD-l-emission data and CD80-emission data.

144. The method of any of claims 123 to 143, further comprising providing a spatial and / or temporal image of the immune checkpoint biomarker in the tissue.

145. The method of claim 144, comprising providing a spatial and / or temporal image of PD-Ll, PD1 and / or CD80 in the tissue.

146. The method of any of claims 123 to 145, comprising determining colocalization of two or more of the immune checkpoint biomarkers in the tissue.

147. The method of claim 146, wherein the method comprises determining:PD1 colocalization with PDL1;CD80 colocalization with PDL1; and / orPD1 colocalization with CD80.

148. The method of any of claim 123 to 147, wherein the subject has adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma, ependymoma, breast cancer, carcinoid tumor, carcinoma of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewings family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell cancer), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell and small cell, lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, salivary gland cancer, sezary syndrome, skin cancer, kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma,malignant, thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

149. The method of any of claim 123 to 147, wherein the subject has melanoma.

150. The method of any of claim 123 to 147, wherein the subject has T-cell lymphoma.

151. The method of any of claim 123 to 147, wherein each of the targeted fluorescent agent(s) and the non-targeted fluorescent agent comprises a non-specific agent selected from the group consisting of soluble proteins (such as soluble PD-1, PD-L1, CD80), antibodies, antibody fragments (such as Fv, Fab, Fab’, and F(ab’)2 fragments), single chain antibodies (such as scFv), single domain antibodies (such as VHH domains), antibody mimetics (such as affibodies and nanobodies), and mixtures thereof.

152. The method of claim 151, wherein the non-targeted fluorescent agent has one or more pharmacokinetic and physical properties similar to the targeted fluorescent agent.

153. The method of claim 151, wherein the targeted fluorescent agent and the non-targeted fluorescent agent comprise antibodies of the same clonal isotype.

154. The method of any of claims 91, 105, 106, and 123 to 153, wherein the targeted fluorescence emission data is corrected by subtracting and normalizing by the non-targeted fluorescence emission data.

155. The method of any of claims 91, 105, 106, and 123 to 153, wherein the targeted fluorescence emission data is corrected using a ratiometric calculation.

156. The method of claim 155, where the ratiometric calculation comprises dividing the targeted fluorescence emission at one or more time points by an intensity corrected untargeted fluorescence emission at the same one or more time point minus one.

157. The method of claim 156, wherein the intensity corrected untargeted fluorescence emission is determined by multiplying the untargeted fluorescence emission by a ratio of targeted fluorescence emission to untargeted fluorescence emission collected from (a) a standard without the biomarker of interest at the same time point, or (b) from an earlier time point where concentrations of the targeted fluorescent agents and the non-targeted fluorescent agents are essentially the same.

158. The method of any of claims 91, 105, 106, and 123 to 153, wherein the targeted fluorescence emission data is corrected by fitting sequences of normalized targeted and untargeted fluorescence emission intensities with a nonlinear least-squares algorithm.

159. The method of any of claims 154 to 158, wherein the spectral unmixing comprises representing the targeted fluorescence emission data and the non-targeted fluorescence emission data as a linear combination of each of the fluorescent agents.

160. The method of claim 159, further comprising performing a least-squares fitting to calculate relative contributions of each of the fluorescent agents.

161. The method of claim 160, wherein the step of acquiring fluorescence emission data comprises acquiring a set of multispectral images of the sample, wherein each of the multispectral images corresponds to a different wavelength band or range.

162. The method of claim 160 or 161, further comprising generating a three-dimensional (m x n x 1) image cube from each acquisition of fluorescence emission data, where m is the number of row-pixels, n is the number of column-pixels, and 1 is the number of wavelengths.

163. The method of claim 162, wherein a measured signal intensity y(^)atwavelength X for each of the fluorescent agents at each pixel of each of the multispectral images is determined by the formula:where x is the relevant amount of the fluorescent agent i, for M different fluorophores that is output from the least-squares fitting; and F is the reference fluorescence emission spectrum for the fluorescent agent.

164. The method of any of claims 154 to 163, wherein the sample is illuminated with a broadband lamp filtered through a multiband excitation filter at stimulus wavelength(s) in bands or ranges of from about 535 to about 545 nm, from about 620 to about 645 nm, and from about 740 to about 750 nm.

165. The method of any of claims 154 to 163, wherein the multiband excitation filter comprises passbands of from about 538 to about 543 nm, from about 623 to about 641 nm, and from about 743 to about 747 nm.

166. The method of any of claims 154 to 165, wherein the fluorescence emission data is acquired through a multiband emission filter to obtain narrowband images in 5 nm increments from about 560 to about 860 nm.

167. The method of any of claims 154 to 166, wherein the fluorescence emission data is acquired through a liquid crystal tunable filter.

168. The method of any of claims 154 to 167, wherein the fluorescence emission data is acquired in bands or ranges of from about 555 to about 610 nm, from about 655 to about 730 nm, and from about 760 to about 850 nm.

169. The method of any of claims 154 to 167, wherein the fluorescence emission data is acquired in bands or ranges of from about 557 to about 602, from about 660 to about 722, and from about 768 to about 846 nm.

170. The method of any of claims 154 to 169, wherein each of the targeted fluorescent agents comprises a fluorophore selected from the group consisting of fluorescent dyes having emissionwavelengths of about 550 nm, about 650 nm, about 680 nm, about 700 nm, about 750 nm, and about 800 nm.

171. The method of any of claims 154 to 169, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 550 nm.

172. The method of any of claims any of claims 154 to 169, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 650 nm.

173. The method of any of claims any of claims 154 to 169, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 680 nm.

174. The method of any of claims any of claims 154 to 169, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 700 nm.

175. The method of any of claims any of claims 154 to 169, wherein at least one of the targeted fluorescent agents comprises a fluorophore having an emission wavelength of about 750 nm.

176. The method of any of claims any of claims 154 to 169, wherein an untargeted fluorescent agents comprises a fluorophore having an emission wavelength of about 800 nm.

177. The method of any of claims 154 to 176, further comprising acquiring autofluorescence emission data prior to contacting the sample with the set of targeted fluorescent agents and the non-targeted fluorescent agent, andcorrecting the targeted fluorescence emission data using the autofluorescence emission data.

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