Tumor and immune cell imaging based on PD-L1 expression

Abstract

The presently disclosed subject matter provides compositions, kits, and methods comprising imaging agents capable of detecting programmed death-ligand 1 (PD-L1). The imaging agents of the present disclosure can be used to detect diseases and disorders such as cancer, infection, and inflammation in a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 438,575, filed December 23, 2016, and U.S. Provisional Patent Application No. 62 / 519,534, filed June 14, 2017, which are incorporated herein by reference in their entireties. Federally funded research and development

[0002] This invention was made with government support under NIH R01CA16631 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention. [Background technology]

[0003] Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immunomodulatory therapies. Few imaging agents can rapidly report on the targets of immunomodulatory therapies.

[0004] Immunotherapy, which suppresses a patient's own immune system to kill cancer cells, plays a central role in the treatment of various cancers (Topalian et al., 2016). Despite significantly improved treatment outcomes, many cancers do not respond to immunomodulatory therapies. Existing companion diagnostics, which work via immunohistochemistry (IHC), provide only a snapshot of the dynamic tumor immune environment and often do not accurately predict treatment response (Mansfield and Dong, 2016). Noninvasive imaging techniques can provide quantitative, real-time assessments of tumor biology and guide drug development (Willmann et al., 2008).

[0005] Positron emission tomography (PET), the most molecular and quantitative translational imaging technique, has been used for repeated measurements of global target expression in all lesions of a given patient to detect estrogen receptor (ER)-positive breast cancer. 18 F] fluoroestradiol ( 18Molecularly targeted PET tracers such as F-FES can predict response to treatment and progression-free survival (Peterson et al., 2008 and Linden et al., 2006). PET tracers, as well as contrast agents for other imaging methodologies, including but not limited to magnetic resonance imaging (MRI), fluorescence imaging, near-infrared (NIR) imaging, photoacoustic imaging, and Raman imaging, can provide rapid, real-time assessment of target expression relevant to immunomodulatory therapy, which may be of great benefit to ongoing clinical trials.

[0006] Programmed death-ligand 1 (PD-L1) is an immune checkpoint protein overexpressed in several cancers and contributes to tumor immunosuppression. Tumor PD-L1 expression indicates tumor response to PD-1 and PD-L1-targeted therapies. It has been shown that radiolabeled anti-PD-L1 antibodies can be used to noninvasively assess PD-L1 expression in human tumor xenografts and syngeneic tumor models (Heskamp et al., 2015; Maute et al., 2015; Chatterjee et al., 2016; Deng et al., 2016; Hettich et al., 2016; Josefson et al., 2016). Radiolabeled antibody conjugates are increasingly being used for imaging tumor-specific proteins, but they require long clearance times of up to several days to improve contrast and lesion detection (Pandit-Taskar et al., 2015; Oosting et al., 2016). Summary of the Invention

[0007] In some embodiments, the presently disclosed subject matter provides imaging agents comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, when present, connects the peptide and the reporting moiety, or, when the linker is absent, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide. In other embodiments, the reporting moiety is incorporated directly into the peptide, for example, where the reporting moiety comprises a radiolabeled amino acid of the peptide, such as a radiolabeled iodotyrosine or fluorotyrosine.

[0008] In a particular embodiment, the peptide that has binding specificity for PD-L1 interacts with amino acids Y56, E58, A113, M115, and Y123 of PD-L1.

[0009] In certain embodiments, the peptide is WL12 and the imaging agent is a compound selected from the group consisting of Formula (I), Formula (II), and Formula (III): [ka] DK-A-221-(L) n -Rpt (II); or DK-A-222-(L) n -Rpt (III); wherein n is an integer selected from the group consisting of 0 and 1; L is a linker; and Rpt is a reporting moiety; and wherein the reporting moiety or linker, if present, is attached to a primary amine group of an amino acid of a peptide comprising an imaging agent of Formula (I), Formula (II), or Formula (III).

[0010] In certain embodiments, the compound of formula (I) is WL12 DOTA: [ka]

[0011] In another aspect, the presently disclosed subject matter provides an imaging method for detecting programmed death-ligand 1 (PD-L1), comprising: (a) providing an effective amount of an imaging agent comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, when present, connects the peptide and the reporting moiety, or, when not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide; (b) contacting one or more cells or tissues with the imaging agent; and (c) generating an image to detect PD-L1. In certain aspects, the imaging agent is a compound of Formula (I) or a peptide that interacts with Y56, E58, A113, M115, and Y123 of PD-L1.

[0012] In certain aspects, the imaging agents of the present disclosure can be used to detect diseases and disorders such as cancer, infection, and inflammation in a subject.

[0013] In still a further aspect, the presently disclosed subject matter provides a kit for detecting programmed death-ligand 1 (PD-L1), the kit comprising an imaging agent comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, when present, links the peptide and the reporting moiety, and wherein, when the linker is not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide.

[0014] While certain aspects of the presently disclosed subject matter have been set forth above and are covered in whole or in part by the presently disclosed subject matter, other aspects will become apparent as the description proceeds in connection with the accompanying examples and figures, as best described herein below. [Brief explanation of the drawings]

[0015] Having thus described the subject matter of the present disclosure in general terms, reference is now made to the accompanying drawings, which are not necessarily drawn to scale: [Figure 1] Figure 1A shows WL12 binding to PD-L1. Figure 1A shows structural diagrams of WL12 and its analogs, as well as the amino acid sequence of WL12 (WL12 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Leu-Hyp-Trp-Ser-Trp(methyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2). Figure 1B shows the predicted binding mode of WL12 to PD-L1. WL12 forms a beta-sheet-like structure within the PD-L1 groove. WL12 is shown in cyan. A surface diagram of PD-L1 is shown in gray, with ribbons and major side chains in magenta; Figure 1C shows that WL12 mimics the binding of PD-1 to PD-L1. The PD-1 structure is shown in cyan. The two major interacting beta strands of PD-1 overlap well with the conformation adopted by WL12 bound to PD-L1; [Figure 2] FIG. 1 shows the far-UV CD spectrum of peptide WL12. [Figure 3] Figure 1 shows the electrospray ionization (ESI) mass spectrum of WL; Theoretical formula: C91H128N22O20S2. Found m / z: 1882.7-(M+1)+1, 941.9-(M+2)+2 / 2. Expected: 1882.19; [Figure 4] Figure 1 shows RP-HPLC purification of WL12D; [Figure 5] Figure 1 shows the low-resolution mass spectrum of PDL1-PD; theoretical formula: C91H128N22O20S2, exact mass: 2339.14, molecular weight: 2340.65, observed m / z: 2340.9-(M+1)+1, 1171.1-(M+2)+2 / 2 and 781.1-(M+2)+3 / 3; [Figure 6] Figure 1 shows RP-HPLC purification of [PDL1-PD-Cu2+]; [Figure 7]Figure 1 shows the low-resolution mass spectrum of the [PDL1-PD-Cu2+] complex; theoretical formula: C110H156N26O29S, exact mass: 2400.05, molecular weight: 2402.18, observed m / z: 2402.6-(M+1)+1, 1201.9-(M+2)+2 / 2; [Figure 8] Figure 8 shows in vitro characterization of the PD-L1 binding peptide WL12; Figure 8A shows a competitive inhibition assay demonstrating the affinity of the WL12 analog for inhibiting the PD-1:PD-L1 interaction; Figure 8B shows flow cytometry histograms of cell lines used in the in vitro study that exhibit variable PD-L1 expression; Figure 8C shows that [64Cu]W12 demonstrates increased binding to cells with high PD-L1 expression that can be blocked by excess peptide (PEP); [Figure 9] Figure 1 shows a representative curve of PD-L1 binding to PD-1; KD = 69.66 ± 11.65 nM (95% CI 44.82-94.48 nM); [Figure 10] Figure 1 shows a representative curve for inhibition of PD-L1 binding to PD-1 using WL12D-Cu2+ complex; IC50=2.97nM (95%CI 2.17~40.5nM) Ki=1.38nM (95%CI 1.01~1.89nM); [Figure 11] Figure 1 shows RP-HPLC chromatograms of [64Cu]WL12 radioactive tracer (red) and the "non-radioactive" WL12-Cu2+ complex; [Figure 12] Figure 1 shows the RP-HPLC chromatogram of the mixture of PDL1-PD and [PDL1-PD-Cu 2+ ]; [Figure 13] Figure 1 shows the mean fluorescence intensity values ​​of the various cell lines used in the uptake assay; [Figure 14] Figure 1 shows the correlation of cell line MFI vs. %ID; [Figure 15]Figure 15 shows rapid in vivo detection of tumor PD-L1 expression using [64Cu]WL12; NSG mice bearing hPD-L1 (red arrow) and CHO tumors (blue arrow) were intravenously administered 150 μCi of [64Cu]WL12, and images were acquired 10, 30, 60, and 120 minutes after radiotracer injection. Figure 15A shows cross-sectional (top) and 3D volume-rendered (bottom) images demonstrating specific accumulation of [64Cu]WL12 in hPD-L1 tumors; Figure 15B shows PD-L1 IHC demonstrating strong immunoreactivity (brown) in hPD-L1 tumors; [Figure 16] Figure 1 shows the specific uptake of [64Cu]WL12 in hPD-L1 tumors in NSG mice; representative volume-rendered PET-CT images of NSG mice bearing hPD-L1 and CHO tumors and injected with [64Cu]WL12 24 hours after tracer injection. Increased uptake in hPD-L1 (red arrow) tumors compared with CHO (blue arrow) tumors confirms PD-L1-mediated uptake of the radiotracer; [Figure 17] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in NSG mice bearing hPD-L1 and CHO tumors. NSG mice were intravenously administered 20 μCi of [64Cu]WL12, and tissues were harvested 60 and 120 minutes after injection. For blocking studies, mice were given an excess of peptide (pep) in a radiotracer injection. [Figure 18] Figure 18 shows: (Figure 18A) Structure of W112-IR800CW conjugate (chemical formula: C137H177N24O34, molecular weight: 2864.34); (Figure 18B) HPLC chromatogram of WL12-IR800CW with UV-Vis spectrum recorded under the peak indicating the conjugation of the dye with the peptide (inset); (Figure 18C) ESI-MS spectrum of WL12-IR800CW conjugate, correlating with the expected molecular weight; [Figure 19]Figure 19 shows evaluation of WL12-IR800CW in CHO and hPDL1 tumor-bearing mice: (Figure 19A) Representative images of mice and ex vivo organs injected with 5 nmol of WL12-IR800CW recorded 24 h after conjugate injection; (Figure 19B, blocking) Representative images of mice injected with 25 nmol of unmodified WL12 and 5 nmol of WL12-IR800CW acquired at 24 h pi; (Figure 19C) Quantification of ex vivo biodistribution of WL12-IR800CW in selected organs and tumors from mice treated with 1 nmol, 3 nmol, and 5 nmol of conjugate and blocked with WL12 (numbers indicate corresponding organs, n=4); [Figure 20] Figure 1: [111In]atezolizumab uptake in human NSCLC and TNBC xenografts is not entirely expression-dependent. (A) Flow cytometry analysis of various TNBC and NSCLC cell lines showing variable PD-L1 expression; (B) [111In]AtzMab binding to cancer cell lines is PD-L1 expression-dependent; (C) Increased [111In]AtzMab uptake in PD-L1-high MDAMB231 TNBC xenografts compared to PD-L1-low SUM149; (D) Increased [111In]AtzMab uptake in PD-L1-high H2444 NSCLC xenografts compared to PD-L1-low H1155. Corresponding histology is shown. From Chatterjee et al., Oncotarget, 2016; [Figure 21]Figure 1 shows that [64Cu]WL12-PET detects AtzMab accumulation in tumors; (A) Whole-body [64Cu]WL12 images show specific accumulation of radioactivity in hPD-L1 tumors at 60 minutes post-tracer injection; (B) [64Cu]WL12 uptake is significantly reduced in hPD-L1 tumors in mice administered a 20 mg / kg dose of AtzMab 24 hours prior to tracer injection; (C) Corresponding biodistribution studies confirmed the feasibility of [64Cu]WL12 to detect AtzMab PD-L1 engagement in tumors; (D) WL12 inhibits AtzMab binding to PD-L1. hPD-L1 cells incubated with serial dilutions of WL12 were stained with Cy5-AtzMab or the commercially available BD antibody BD-MIH1-PE. Mean fluorescence intensity (MFI) versus peptide concentration plots show IC50s of 2.5 nM and 37.8 nM for Cy5-AtzMab and BD-MIH1-PE, respectively; [Figure 22] Figure 1 shows that [64Cu]WL12-PET detects AtzMab accumulation in triple-negative breast cancer xenografts; [64Cu]WL12 uptake is significantly reduced in MDAMB231 tumors in mice administered a 20 mg / Kg dose of AtzMab 24 hours before tracer injection; [Figure 23] Figure 1 shows: (A) Structure of Wl2-IR800 conjugate (chemical formula: C137H177N24O34, molecular weight: 2864.34); (B) HPLC chromatogram of WL12-IR800 with UV-Vis spectrum recorded under the peak indicating the conjugation of the dye with the peptide (inset); (C) ESI-MS spectrum of WL12-IR800, correlating with the expected molecular weight; [Figure 24]Figure 1 shows the evaluation of WL12-IR800 in CHO and hPDL1 tumor-bearing mice; (A) Representative images of mice injected with 5 nmol of WL12-IR800 and ex vivo organs recorded 24 h after conjugate injection; (B, blocking) Representative images of mice injected with 25 nmol of unmodified WL12 and 5 nmol of WL12-IR800 acquired at 24 h pi; (C) Quantification of ex vivo biodistribution of WL12-IR800 in selected organs and tumors from mice treated with 1 nmol, 3 nmol, and 5 nmol of conjugate and blocked with WL12 (numbers indicate corresponding organs, n=4); [Figure 25] Figure 1: Evaluation of [68Ga]WL12 in CHO and CHO-hPDL1 tumor models. (A) PET-CT (volume-rendered) images of [68Ga]WL12 uptake in CHO-hPDL1 (red arrow, high PD-L1 expression) and CHO (black arrow, low PD-L1 expression) tumors (n=3) confirm PD-L1-mediated uptake of the radiotracer. (B) Ex vivo biodistribution analysis 1 hour after injection of [68Ga]WL12 in the same tumor model. Blocking dose cohorts were co-injected with 50 micrograms of non-radioactive peptide. [Figure 26] Figure 1 shows the evaluation of [18F]WL12 in CHO and CHO-hPDL1 tumor models; (A) PET-CT (volume-rendered) images of [18F]WL12 uptake in CHO-hPD-L1 (red arrow, high PD-L1 expression) and CHO (blue arrow, low PD-L1 expression) tumors (n=3) confirming PD-L1-mediated uptake of the radiotracer; [Figure 27] Figure 1 shows that mice bearing MDAMB231 and SUM149 tumors were injected with a 20 mg / Kg dose of atezolizumab; 20 hours after mAb administration, the mice were injected with 20 μCi of [64Cu]WL12, and biodistribution studies were performed 24 hours after tracer injection. The data demonstrate that atezolizumab binding to PD-L1 in tumors can be quantified by [64Cu]WL12; [Figure 28] Figure 1 shows dose-dependent PD-L1 occupancy determination for the PD-L1 therapeutic antibody atezolizumab. Mice bearing MDAB231 breast tumors were injected with various doses of atezolizumab, and 24 hours later, the mice were injected with [64Cu]WL12, and biodistribution studies were performed 2 hours after tracer injection. The data show that [64Cu]WL12 accumulation in the tumors decreased with increasing antibody dose; [Figure 29] Figure 1 shows the time- and dose-dependent changes in PD-L1 occupancy of atezolizumab as measured by [64Cu]WL12. MDAMB231 tumor-bearing mice were administered atezolizumab at a dose of 1 or 10 mg / kg. At 24 or 120 hours after mAb administration, the mice were injected with [64Cu]WL12, and tumor accumulation of radioactivity was measured by biodistribution studies. As expected, complete PD-L1 blockade was observed at both 24 and 120 hours with the 10 mg / kg dose. With the 1 mg / kg dose, increased accumulation of [64Cu]WL12 was observed at 120 hours but not at 24 hours, suggesting efflux of atezolizumab from the tumor over time when low mAb doses are used. These data suggest that the peptides of the present disclosure can be used to analyze PD-L1 therapeutic mAb residence time in tumors; [Figure 30] 1 shows the chemical structures of DK-A-221 and DK-A-222; [Figure 31] Figure 1 shows data for the DK222 PD-L1 binding peptide. NOTA-conjugated DK222 was synthesized and evaluated in CHO / CHO-HPD-L1 tumor-bearing mice. Imaging (A) and biodistribution (B) data demonstrate excellent pharmacokinetics of [64Cu]DK222; [Figure 32] Figure 1 shows the biodistribution of [64Cu]DK222 in NSG mice bearing CHO / CHO-hPD-L1 tumors; [Figure 33]Figures 33A and 33B demonstrate that WL12 inhibits the interaction between PD-1 and PD-L1 therapeutics in vitro. Figure 33A shows that the binding mode of WL12 to PD-L1 (green and cyan) overlaps with the binding mode of PD-1 to AtzMab (red and cyan), AveMab (orange and cyan), and DurMab (blue and cyan). Non-interacting residues are shown in gray. The diverse contacts encompassing the common binding region (cyan) explain the diverse binding mechanisms of different therapeutic mAbs. Figure 33B shows that WL12 inhibits Cy-5-conjugated AtzMab, AveMab, and DurMab to PD-L1, as demonstrated through competitive inhibition. Mean fluorescence intensity was determined by flow cytometry. Figure 33C shows that [64Cu]WL12 binding to PD-L1-positive HCC827, H226, hPD-L1, and MDAMB231 cells is inhibited in the presence of 60 nM AtzMab, AveMab, and DurMab compared to the PBS control. [64Cu]WL12 binding in PD-L1-negative CHO and SUM149 cells is also shown. ****, P<0.0001; NS, non-significant; [Figure 34](Figure 34A) Diagram of the molecular surface surrounding the PD-L1 interaction interface with PD-1. Common residues involved in interactions with PD-1 competitive therapeutics are shown in cyan, molecular contacts specific to PD-1 interactions are shown in purple, and non-interacting residues are shown in gray. To illustrate the overlap of intermolecular interactions, the structure of bound PD-1 is shown in purple, and the predicted conformation of WL12 is shown in green; (Figure 34B) WL12 inhibits the binding of Cy5-conjugated PD-1-Fc protein to PD-L1 in hPD-L1 cells. Mean fluorescence intensity determined by flow cytometry; (Figure 35C) WL12 (5 nM) inhibits the binding of Cy5-conjugated AtzMab, AveMab, and DurMab (2 nM) to PD-L1 in HCC827 and H226 cells. Mean fluorescence intensity measured by flow cytometry and Figure 35D shows the mean fluorescence intensity determined by flow cytometry from Figure 34B and Figure 35C; [Figure 35] Figures 35A-H demonstrate that PD-L1 engagement by PD-L1 mAbs is quantified in tumors using [64Cu]WL12 in xenografts with variable PD-L1 expression. Figures 35A-H show reduced uptake of [64Cu]WL12 in H226 (Figures 35A, 35B), HCC827 (Figures 35C, 35D), and hPD-L1 / CHO (Figures 35G, 35H) xenografts in mice treated with 20 mg / kg AtzMab 24 hours prior to radiotracer injection compared to saline-treated controls. Whole-body, volume-rendered [64Cu]WL12 PET-CT images (Figures 35A, 35D, 35G) and ex vivo biodistribution (Figures 35B, 35E, 35H). Figures 35C, 35F, and 35I show IHC staining for PD-L1 from corresponding tumors. ****, P<0.0001. ***, P<0.001; NS, non-significant; [Figure 36]Figures 36A, PD-L1 expression in various cell lines and corresponding mean fluorescence intensity values. Figures 36B, 36C, and 36D, ex vivo biodistribution of [64Cu]WL12 in tumor-bearing mice with H226 (B), HCC827 (C), or hPD-L1 / CHO (D) tumors administered a 20 mg / Kg dose of AtzMab 24 hours prior to tracer injection. Data shown are mean ± SEM. ****, P<0.0001; ***, P<0.001; NS, non-significant; [Figure 37] Figure 37 demonstrates the dynamic changes in tumor PD-L1 expression detected with [64Cu]WL12 and its engagement by AtzMab. Figure 37A shows the increase in PD-L1 cell surface expression in A549-iPDL1 cells treated with doxycycline for 6 and 72 hours. Flow cytometry histogram. Figure 37B shows that WL12 (5 nM) inhibits the binding of Cy5-conjugated AtzMab, AveMab, and DurMab (2 nM) to A549-iPD-L1 cells treated with doxycycline for 72 hours. Figure 37C shows that [64Cu]WL12 binding to A549-iPDL1 cells (72 hours doxycycline) is significantly reduced in the presence of 60 nM AtzMab compared to control. Figures 37D and 37E show that [64Cu]WL12 uptake in A549-iPDL1 xenografts was significantly lower in mice that received intravenous AtzMab 24 hours prior to radiotracer injection compared with saline controls, and similar to parental A549 xenografts. Volume-rendered whole-body PET-CT images (D) and ex vivo quantification (Figure 37E). Figure 37F shows IHC staining for PD-L1 in the corresponding tumors. ****, P<0.0001; NS, non-significant; [Figure 38] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in A549-iPDL1 and A549 control tumor-bearing mice administered doxycycline for 72 hours and treated with 20 mg / Kg AtzMab 24 hours before radiotracer injection. ****, P<0.0001; NS, non-significant; [Figure 39]Figures 39A-E demonstrate tumor PD-L1 engagement by three different PD-L1 therapeutic mAbs quantified with [64Cu]WL12. Uptake of [64Cu]WL12 in MDAMB231 xenografts is significantly reduced in mice administered AtzMab (20 mg / kg), AveMab (10 mg / kg), or DurMab (10 mg / kg) 24 hours prior to radiotracer injection. Whole-body volume-rendered [64Cu]WL12 PET-CT images and ex vivo biodistribution (Figure 39E) of saline (Figure 39A), AtzMab (Figure 39B), AveMab (Figure 39C), and DurMab (Figure 39D)-treated mice. Figure 39F shows IHC staining of PD-L1 in the corresponding tumors. ****, P<0.0001; NS, non-significant. [Figure 40] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in MDAMB231-bearing mice treated with AtzMab (20 mg / Kg), AveMab (10 mg / Kg), or DurMab (10 mg / Kg) 24 hours before radiotracer injection. ****, P<0.0001; NS, non-significant; [Figure 41]Figure 41 demonstrates the effect of dose and time on tumor PD-L1 occupancy by AtzMab quantified using [64Cu]WL12. Figure 41A shows the dose-exposure relationship demonstrating the reduction of free PD-L1 ligand in MDA-MB-231 tumors in mice with increasing AtzMab dose (mg / kg). Whole-body [64Cu]WL12 PET-CT images of MDA-MB-231 tumor-bearing mice administered 0.06 mg / kg, 0.6 mg / kg, and 3.2 mg / kg AtzMab (Figure 41A). Figures 41B and 41C show ex vivo quantification of [64Cu]WL12 uptake in tumors of mice treated with increasing doses of AtzMab (0.0009-24 mg / kg). AtzMab was injected 24 hours before radiotracer injection (Figure 41B). The percentage of free PD-L1 ligand relative to the median free PD-L1 ligand measured at 0 mg / kg was calculated (Figure 41C). Blue open circles: free PD-L1 ligand measured for each dose level in mice. Red dashed line: average model-predicted dose-response relationship. Figures 41D and 41E show the AtzMab (mg / kg) dose effect on tumor PD-L1 occupancy over time, showing an increase in free PD-L1 ligand at AtzMab doses of 0.6 or 1 mg / kg, but not at AtzMab doses of 10 or 20 mg / kg, summarizing the nonlinear kinetics of the mAb. Whole-body volume-rendered [64Cu]WL12 PET-CT images (D) and ex vivo biodistribution (E). ****, P<0.0001; NS, non-significant; [Figure 42] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in MDAMB231 tumor-bearing mice at increasing doses of AtzMab (0.0009–12 mg / Kg) 24 h before tracer injection; [Figure 43] The structural diagrams of DK-A-221 and DK-A-222 and their analogs, as well as the amino acid sequence of DK-A-221, are shown (DK-A-221 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2).

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. DETAILED DESCRIPTION OF THE INVENTION

[0017] The presently disclosed subject matter is described more fully below with reference to the accompanying drawings, in which some, but not all, embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it should be understood that the presently disclosed subject matter is not to be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0018] I. Compositions Comprising Contrast Agents In some embodiments, the presently disclosed subject matter provides highly specific peptide-based positron emission tomography (PET) imaging agents for detecting immune checkpoint proteins such as PD-L1. These imaging agents can be used to detect tumor PD-L1 expression specifically and immediately after administration to a subject.

[0019] Thus, in some embodiments, the presently disclosed subject matter provides imaging agents comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, where the linker, when present, connects the peptide and the reporting moiety, or, when the linker is absent, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide. In other embodiments, the reporting moiety is incorporated directly into the peptide, for example, where the reporting moiety comprises a radiolabeled amino acid of the peptide, such as a radiolabeled iodotyrosine or fluorotyrosine.

[0020] In some embodiments, peptides that have binding specificity for programmed death-ligand 1 (PD-L1) may interact with four specific amino acids of PD-L1. In certain embodiments, the peptides may interact with amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, peptides that have binding specificity for PD-L1 may interact with five specific amino acids of PD-L1. In certain embodiments, the peptides may interact with amino acids Y56, E58, A113, M115, and Y123 of PD-L1. In some embodiments, the peptide that interacts with PD-L1 is peptide WL12. Peptide WL12 may have the amino acid sequence of cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Leu-Hyp-Trp-Ser-Trp(methyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2) (SEQ ID NO: 1). In some embodiments, WL12 may interact with four amino acids of PD-L1. In specific embodiments, WL12 may interact with amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, WL12 may interact with five amino acids of PD-L1. In specific embodiments, WL12 may interact with amino acids Y56, E58, A113, M115, and Y123 of PD-L1. In other embodiments, the peptide that interacts with PD-L1 is DK-A-221. Peptide DK-A-221 may have the amino acid sequence of cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2) (SEQ ID NO: 2). In some embodiments, DK-A-221 can interact with four amino acids of PD-L1. In specific embodiments, DK-A-221 can interact with amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, DK-A-221 can interact with five amino acids of PD-L1. In specific embodiments, DK-A-221 can interact with amino acids Y56, E58, A113, M115, and Y123 of PD-L1.In other embodiments, the peptide that interacts with PD-L1 is DK-A-222. In some embodiments, DK-A-222 can interact with four amino acids of PD-L1. In specific embodiments, DK-A-222 can interact with amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, DK-A-222 can interact with five amino acids of PD-L1. In specific embodiments, DK-A-222 can interact with amino acids Y56, E58, A113, M115, and Y123 of PD-L1.

[0021] In some embodiments, peptides that have binding specificity for PD-L1 may have at least 80% sequence identity to SEQ ID NO: 1. Peptides that have binding specificity for PD-L1 may have at least 80% sequence identity to SEQ ID NO: 2. Peptides that have binding specificity for PD-L1 may have at least 85% sequence identity to SEQ ID NO: 1. Peptides that have binding specificity for PD-L1 may have at least 85% sequence identity to SEQ ID NO: 2. Peptides that have binding specificity for PD-L1 may have at least 90% sequence identity to SEQ ID NO: 1. Peptides that have binding specificity for PD-L1 may have at least 90% sequence identity to SEQ ID NO: 2. Peptides that have binding specificity for PD-L1 may have at least 95% sequence identity to SEQ ID NO: 1. Peptides that have binding specificity for PD-L1 may have at least 95% sequence identity to SEQ ID NO: 2. Peptides that have binding specificity for PD-L1 may have 100% sequence identity to SEQ ID NO: 1. A peptide that has binding specificity for PD-L1 may have 100% sequence identity to SEQ ID NO:2.

[0022] The term "percent identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be easily calculated by known methods, including but not limited to those described in the following: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Madison, Wis.).Multiple alignment of sequences can be performed using the Clustal alignment method (Higgins and Sharp (1989) CABIOS. 5:151-153) using default parameters, including default parameters for pairwise alignment.

[0023] As used herein, the terms "amino acid" and "residue" are interchangeable and, when used in the context of a peptide or polypeptide, refer to naturally occurring and synthetic amino acids, as well as amino acid analogs, amino acid mimetics, and non-naturally occurring amino acids that are chemically similar to naturally occurring amino acids.

[0024] The terms "naturally occurring amino acid" and "naturally encoded amino acid" are used interchangeably and refer to amino acids encoded by the genetic code, as well as amino acids encoded by the genetic code that are modified after synthesis, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

[0025] An "amino acid analog" is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a compound that has an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but will retain the same basic chemical structure as a naturally occurring amino acid.

[0026] The terms "non-naturally occurring amino acid" and "non-naturally encoded amino acid" are used interchangeably and refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, but that are not incorporated into a growing polypeptide chain by the translation complex. "Non-naturally occurring amino acids" also include, but are not limited to, amino acids that arise by modification (e.g., post-translational modification) of a naturally encoded amino acid (including, but not limited to, the 20 standard amino acids), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. A non-limiting list of examples of non-naturally occurring amino acids that can be inserted into or substituted for wild-type residues in a polypeptide sequence includes β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. Examples include (in L- or D-form; abbreviated in parentheses): citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMcCit), Nα-methylhomocitrulline (Nα-MeHoCit), or ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeHoK).Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-amino-Phe), 4 -guanidinophenylalanine (Guf), glycyrrhizin (abbreviated as "K(Nε-glycyl)" or "K(glycyl)" or "K(gly)"), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methylvaline (NMeVal), Nα-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocapric acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp).γ-carboxyglutamate, ε-N,N,N-trimethyllysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino-O-phthalic acid (4APA), N-acetylglucosaminyl-L-serine, N-acetylglucosylaminyl-L-threonine, O-phosphotyrosine and other similar amino acids, as well as derivatized forms of any of those specifically listed.

[0027] A "peptide" or "protein" comprises a series of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. A peptide may refer to an individual peptide or a collection of peptides. One or more amino acids in the imaging agents of the present disclosure may also be modified, for example, by the addition of chemicals such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, sulfoxide groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. In some embodiments, other modifications may include the incorporation of D-amino acids, conjugation of other molecules at the N- and C-termini, conjugation of biomolecules such as fluorescent probes, poly(ethylene glycol), targeting ligands, retroinversion, etc. Any modification should not substantially interfere with the desired biological activity of the peptide.

[0028] In some embodiments of the imaging agents of the present disclosure, the reporting moiety is selected from the group consisting of a chelator, a radiolabeled substrate, a fluorescent dye, a photoacoustic reporting molecule, and a Raman-active reporting molecule.

[0029] In some embodiments of the imaging agent of the present disclosure, the reporting moiety is a chelator, and the chelator is selected from the group consisting of: DOTAGA (1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTASA (1,4,7,10-tetraazacyclododecane-1-(2-succinic acid)-4, 7,10-triacetic acid), CB-DO2A (10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tetraazabicyclo[5.5.2]tetradecane]), Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), TCMC (2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)-cyclododecane), Oxo-DO3A (1-oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-tri- acetic acid), p-NH2-Bn-oxo-DO3A (1-oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((1,8--N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane)), MM-TE2A, DM-TE2A, CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid), CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODA (1,4,7-triazacyclononane-1,4-diacetate); NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), (NOTAGA) 1,4,7-triazonane-1,4- diyl)diacetic acid DFO (desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-TM (-N,N',N',tris(2-mercaptoethyl)-1,4,7-triazacyclononane), Diamsar (1,8-diamino-3,6,10,13,16,19-hexazabicyclo(6,6,6)eicosane, 3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), Sarar (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6] ]eicosane-1,8-diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19-hexazabicyclo[6.6.6]icosan-1-ylamino)methyl)benzoic acid), and BaBaSar.

[0030] In some embodiments, peptide, linker, reporter conjugate are prepared by click chemistry.See, for example, International Patent Application Publication WO / 2017 / 027870 to Pomper et al., for triazole-conjugated urea, thiourea, carbamate, and "reverse" carbamate for PSMA-targeted imaging agent and its use, published on February 16, 1997, and US Patent Application Publication No. 20140341804 to Pomper et al., for homo- and hetero-multivalent inhibitors of prostate-specific membrane antigen (Pmsa) and its use, published on November 20, 2014, each of which is incorporated by reference in its entirety.

[0031] In certain embodiments, the chelator has a structure selected from the following: [ka]

[0032] In a more particular embodiment, the reporting moiety is a chelator, and the chelator is 94m Tc, 99m Tc, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 55 Co, 57 Co, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 153 Sm, 166 Ho, 152 Gd, 82 Rb, 89 Zr, and 166 It further comprises a radioactive metal selected from the group consisting of Dy.

[0033] In other embodiments of the imaging agent of the present disclosure, the reporting moiety is a radiolabeled substrate, the radiolabeled substrate comprising: 11 C. 13 N, 15 O. 123 I, 124 I, 125 I, 126 I, 131 I, 75 Br, 76 Br, 77 Br, 80 Br, 80m Br, 82 Br, 83 Br, and 211 In certain embodiments, the radiolabeled substrate comprises a 18F-labeled substrate. In even more particular embodiments, the radiolabeled substrate comprises a radioisotope selected from the group consisting of: At 18 The F-labeled substrate is selected from the group consisting of 2-fluoro-PABA, 3-fluoro-PABA, 2-fluoro-mannitol, and N-succinimidyl-4-fluorobenzoate. In some embodiments, the substrate is labeled using the AlF method, for example, based on the chelation of aluminum fluoride with NOTA, NODA, or any other suitable chelating agent known in the art. 18 Labeled with F. See, e.g., Liu S., et al., "One-step radiosynthesis of 18 F-AlF-NOTA-RGD2 for tumor angiogenisis PET imaging. Eur J Nucl Med Mol Imaging. 2011, 38(9):1732-41; McBride WJ, et al., “A novel method of 18F radiolabeling for PET. J Nucl Med. 2009;50:991-998; McBride WJ, D'Souza CA, Sharkey RM, Sharkey RM, Karacay H, Rossi EA, Chang CH, Goldenberg DM. Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex. Bioconjug Chem. 2010;21:1331-1340.

[0034] In other embodiments of the imaging agent of the present disclosure, the reporting moiety is a fluorescent dye, and the fluorescent dye is selected from the group consisting of: carbocyanine, indocarbocyanine, oxacarbocyanine, zicarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY) dyes, or derivatives thereof, BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY 581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665, including but not limited to Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IR800 (dimethyl{4-[1,5,5-tris(4-dimethylaminophenyl)-2,4-pentadienylidene]-2,5-cyclohexadien-1-ylidene}ammonium perchlorate), IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0035] In other embodiments of the imaging agent of the present disclosure, the reporting moiety is a photoacoustic reporting molecule, and the photoacoustic reporting molecule is selected from the group consisting of a dye or a nanoparticle. In certain embodiments, the dye comprises a fluorescent dye. In even more specific embodiments, the fluorescent dye is selected from the group consisting of indocyanine green (ICG), Alexa Fluor 750, Evans Blue, BHQ 3, QXL 680, IRDye 880CW, MMPSense 680, methylene blue, PPCy-C8, and Cypate-C18. See Wu et al., Int. J. Mol. Sci., 15, 23616-23639 (2014).

[0036] In other embodiments, the nanoparticles are selected from the group consisting of plasmonic nanoparticles, including but not limited to gold nanospheres, gold nanoshells, gold nanorods, gold nanocages, gold nanostars, and gold nanoclusters, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd2O3 nanoparticles, single-walled carbon nanotubes, dye-loaded perfluorocarbon nanoparticles, and superparamagnetic iron oxide nanoparticles.

[0037] In other embodiments of the imaging agent of the present disclosure, the reporting moiety is a Raman-active reporting molecule, and the Raman-active reporting molecule is selected from the group consisting of single-walled carbon nanotubes (SWNTs) and surface-enhanced Raman scattering (SERS) agents. In certain embodiments, the SERS agent comprises a metal (e.g., gold or silver) nanoparticle labeled with a Raman-active reporter molecule. In even more specific embodiments, the Raman-active reporter molecule comprises a fluorescent dye. In certain embodiments, the fluorescent dye is selected from the group consisting of Cy3, Cy5, rhodamine, and chalcogenopyrylium dyes.

[0038] In other embodiments of the imaging agent of the present disclosure, the linker is selected from the group consisting of: (a) [ka] wherein Rpt is a reporting moiety; W1 is selected from the group consisting of C1-C6 alkylene, C3-C6 cycloalkylene, and arylene; W2 is -NR 1 -(C=O)-, -NR 1 -(C=S)-, -(C=O)-NR 1 -, -(C=S)-NR 1 -, and -S-, wherein each R 1 are independently H or C1-C4 alkyl; each R2 is independently H or -COOR3, where each R3 is independently H, C1-C6 alkyl, C2-C 12 Aryl or C4-C 16 alkylaryl; b is an integer selected from the group consisting of 0, 1, 2, and 3; d is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; and wherein the wavy line indicates the point of attachment between the linker and the peptide; (b) Rpt-XYZ-W3- wherein Rpt is a reporting moiety; X and Z can each independently be C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 heteroalkyl, C2-C8 heteroalkenyl, C2-C8 heteroalkynyl, C1-C8 alkoxy, or a bond, each of which may be selected from 0 to 5 R A Y and W3 may each independently be substituted with -O-, -S(O) p -, -NH-, -NR B -, -CH=CH-, -CR B =CH-, -CH=CR B -, -NH-CO-, -NH-CO2-, -NR B -CO-, -NR B -CO2-, -CO-NH-, -CO2-NH-, -CO-NR B -, -CO2-NR B - or a bond; p is 0, 1, or 2; R Ais, at each occurrence, halogen, hydroxy, amino, cyano, nitro, COH, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted mono- or di-alkylamino, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted mono- or di-alkylcarboxamido, optionally substituted aryl, or optionally substituted heteroaryl; R B is, at each occurrence, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted mono- or di-alkylamino, optionally substituted alkylthio, optionally substituted aryl, or optionally substituted heteroaryl; or (c) Amino acid linker.

[0039] In certain embodiments, the imaging agent is a compound selected from the group consisting of Formula (I), Formula (II), and Formula (III): [ka] DK-A-221-(L) n -Rpt(II); or DK-A-222-(L) n -Rpt(III); wherein n is an integer selected from the group consisting of 0 and 1; L is a linker; and Rpt is a reporting moiety; and wherein the reporting moiety or linker, if present, is attached to a primary amine group of a peptide comprising an imaging agent of Formula (I), Formula (II), or Formula (III).

[0040] In certain embodiments, the linker, when present, is 13 Ornithine (Orn) is bound to a primary amine group. In certain embodiments, the reporting moiety comprises a DOTAGA chelator. In more particular embodiments, the DOTAGA chelator is 64Further includes Cu radiometal.

[0041] In a more particular embodiment, the compound of formula (I) is: [ka]

[0042] Those skilled in the art will recognize, upon reviewing the subject matter of the present disclosure, that various combinations of chelating agent / radioactive metal ion are suitable for use with the imaging agents disclosed herein. Representative chelating agents are known in the art. By way of non-limiting example, specific chelating agents and linkers are disclosed in U.S. Patent Application Publication Nos. 2015 / 0246144 and 2015 / 0104387, each of which is incorporated herein by reference in its entirety.

[0043] In some embodiments, the imaging agent is capable of detecting PD-L1 in vitro, in vivo, and / or ex vivo. In some aspects, the imaging agent is capable of detecting PD-L1 in vivo. PD-L1 is expressed by various tumors, and its overexpression is induced in tumor cells as an adaptive mechanism in response to tumor-infiltrating cytotoxic T cells (Topalian et al., 2016). Those skilled in the art will recognize that PD-L1 may contain modifications and / or mutations and still be applicable to the methods of the present disclosure, so long as it can still be detected by the imaging agents of the present disclosure.

[0044] In some embodiments, IC of an imaging agent of the present disclosure that inhibits the interaction of PD-L1 with its ligand, programmed cell death protein 1 (PD-1) 50 In some embodiments, the IC ranges from about 100 nM to about 1 pM. 50 is less than 100 nM, in other embodiments less than 10 nM, in other embodiments less than 8 nM, in other embodiments less than 5 nm, in other embodiments less than 4 nm, and in other embodiments less than 3 nM.

[0045] The term "binding affinity" refers to a property that describes how strongly two or more compounds associate with each other in a non-covalent manner. Binding affinity can be measured qualitatively (such as "strong," "weak," "high," or "low") or quantitatively (such as K d Characterization can be performed (e.g., measurement of

[0046] (II. Detection Methods Using Contrast Agents) In some embodiments, the presently disclosed subject matter provides methods for detecting immune checkpoint proteins such as PD-L1. In some embodiments, the presently disclosed subject matter provides methods for detecting diseases, disorders, or conditions that result in overexpression of PD-L1, such as cancer, inflammation, or infection.

[0047] In some embodiments, the presently disclosed subject matter provides imaging methods for detecting programmed death-ligand 1 (PD-L1), comprising: (a) providing an effective amount of an imaging agent comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, when present, links the peptide and the reporting moiety, or, when the linker is not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide, as described immediately above; (b) contacting one or more cells or tissues with the imaging agent; and (c) generating an image to detect PD-L1.

[0048] As used herein, the term "imaging" or "producing an image" refers to the use of any imaging technique that visualizes a detectable compound by measuring the energy emitted from the compound. In some embodiments, the term "imaging" refers to the use of any imaging technique that visualizes a detectable compound after administration to a subject by measuring the energy emitted from the compound after localization of the compound after administration. In some embodiments, the imaging technique involves administering to the subject a compound that can be detected from outside the subject. In some embodiments, the image is generated by differences in the spatial distribution of an imaging agent that accumulates in various locations in the subject. In some embodiments, the administration of the imaging agent is by injection.

[0049] The term "imaging agent" is intended to include compounds that can be imaged, for example, by positron emission tomography (PET). As used herein, "positron emission tomography imaging" or "PET" includes all positron emission tomography imaging systems or equivalents, and all devices capable of positron emission tomography imaging. The methods of the presently disclosed subject matter can be performed using any such device, or a variation of a PET device or its equivalent, or in conjunction with any known PET methodology. See, for example, U.S. Patent Nos. 6,151,377; 6,072,177; 5,900,636; 5,608,221; 5,532,489; 5,272,343; and 5,103,098, each of which is incorporated herein by reference. Animal imaging modalities include, for example, microPET (Corcorde Microsystems).

[0050] Depending on the reporting moiety, the imaging agents of the present disclosure can be used in PET, single photon emission computed tomography (SPECT), near-infrared (fluorescence), photoacoustic, and Raman imaging.

[0051] In some embodiments, imaging involves scanning the entire subject or patient, or a specific region of the subject or patient, using a detection system to detect a signal. The detected signal is then converted into an image. The resulting image should be interpreted by a skilled observer, such as a physician. Generally, imaging is performed about 1 minute to about 48 hours after administration of the imaging agent. The exact timing of imaging depends on factors such as the clearance rate of the administered compound, as will be readily apparent to those skilled in the art. The imaging time frame may vary based on the radionucleotide used. In certain embodiments, imaging is performed between about 1 minute and about 4 hours after administration, e.g., between 15 and 30 minutes, between 30 and 45 minutes, between 45 and 60 minutes, between 60 and 90 minutes, and between 60 and 120 minutes. In some embodiments, detection of PD-L1 is performed approximately 60 minutes after administration of the imaging agent to the subject. In some embodiments, imaging may be performed 24 hours after injection of the Zr-89-labeled peptide. In some embodiments, imaging may be performed 24 hours after injection of the I-124 labeled peptide.

[0052] Once the image is obtained, one skilled in the art can determine the location of the compound. Using this information, one skilled in the art can determine whether a condition, such as infection, inflammation, or cancer, is present, the extent of the condition, or the effectiveness of a treatment the subject is receiving.

[0053] In some embodiments, contacting of cells or tissues with an imaging agent occurs in vitro, in vivo, or ex vivo. "Contacting" refers to any action that results in at least one imaging agent of the presently disclosed subject matter coming into physical contact with at least one cell or tissue. Thus, it can include exposing a cell(s) or tissue(s) to an imaging agent in an amount sufficient to result in contact between the at least one imaging agent and the at least one cell or tissue. In some embodiments, the method can be performed in vitro or ex vivo by introducing and preferably mixing the imaging agent and cells or tissues in a controlled environment, such as a culture dish or tube. In some embodiments, the method can be performed in vivo, in which case contacting refers to exposing at least one cell or tissue of a subject to at least one imaging agent of the presently disclosed subject matter, such as by administering the imaging agent to the subject via any suitable route. In some embodiments, contacting of cells or tissues with the imaging agent occurs within the subject.

[0054] The term "effective amount" of an imaging agent refers to an amount necessary or sufficient to provide a readable signal when imaged using the techniques described herein, for example, positron emission tomography (PET). The effective amount may vary depending on factors such as the size and weight of the subject, the type of illness, or the specific compound. For example, the choice of compound may affect what constitutes an "effective amount." Those skilled in the art will be able to examine the factors contained herein and make a determination regarding the effective amount of a compound without undue experimentation.

[0055] In many embodiments thereof, the subject diagnosed or treated by the methods of the present disclosure is desirably a human subject, although it should be understood that the methods described herein are effective with respect to all vertebrate species intended to be encompassed by the term "subject." Accordingly, a "subject" can include a human subject for medical purposes, such as for the diagnosis or treatment of an existing disease, disorder, or condition, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include: primates, e.g., mammals, including, but not limited to, humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques, etc.; cattle, e.g., cows, cattle, etc.; ovine, e.g., sheep, etc.; caprine, e.g., goats, etc.; swine, e.g., pigs and adult pigs, etc.; equines, e.g., horses, donkeys, zebras, etc.; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, etc.; and rodents, including mice, rats, guinea pigs, etc. The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Furthermore, a "subject" may include a patient suffering from or suspected of suffering from a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. A subject also includes animal disease models (e.g., rats or mice used in experiments). In some embodiments, the subject is a human, rat, mouse, cat, dog, horse, sheep, cow, monkey, bird, or amphibian.

[0056] In general, the imaging agents of the present disclosure may be administered to a subject for the detection of a disease, disorder, or condition by any suitable route of administration, including: oral, nasal, transmucosal, intraocular, rectal, vaginal, or parenteral, intravenous, intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injection, intracapsular, topical, by powder, ointment, or drops (including eye drops), buccal and sublingual, transdermal, via inhalation spray, or other delivery modes known in the art.

[0057] As used herein, the phrases "systemic administration," "systemic administration," "peripheral administration," and "peripheral administration" refer to administration of compositions such that they enter the system of a subject or patient and are therefore subject to metabolic and other similar processes, e.g., subcutaneous or intravenous administration.

[0058] The phrases "parenteral administration" and "parenteral administration" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0059] In some embodiments, the imaging agents exhibit a target to non-target ratio of at least 3: 1. In some embodiments, the term "target" refers to cells or tissues that exhibit overexpression of PD-L1 protein, and the term "non-target" refers to cells or tissues that do not exhibit overexpression of PD-L1 protein.

[0060] In some embodiments, imaging methods are used to detect cancer. "Cancer" in a subject or patient refers to the presence of cells with characteristics typical of cancer-causing cells, such as uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significantly increased anti-apoptotic activity, rapid growth and proliferation rate, and specific characteristic morphology and cell markers. In some circumstances, cancer cells take the form of a tumor; such cells may exist locally within an animal or circulate in the bloodstream as independent cells, such as leukemia cells. As used herein, cancer includes newly diagnosed or recurrent cancers, including, but not limited to, blastoma, carcinoma, glioma, leukemia, lymphoma, melanoma, myeloma, and sarcoma. As used herein, cancer includes, but is not limited to, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer such as triple-negative breast cancer, prostate cancer, colon cancer, esophageal cancer, gastric cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary tract cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, brain tumor, and adenoma. In some aspects, the cancer comprises stage 0 cancer. In some embodiments, the cancer comprises stage I cancer. In some aspects, the cancer comprises stage II cancer. In some embodiments, the cancer comprises stage III cancer. In some embodiments, the cancer comprises stage IV cancer. In some embodiments, the cancer is refractory and / or metastatic.

[0061] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. As used herein, a "solid tumor" is an abnormal mass of tissue that typically does not contain cysts or liquid areas. Solid tumors may be present in, by way of non-limiting example, the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovaries, cervix, stomach, colon, rectum, bladder, uterus, testes, and pancreas. In some embodiments, imaging methods are used to detect solid tumors. In yet other embodiments, imaging methods are used to detect metastatic cancer.

[0062] In some embodiments, the imaging method is used to detect infectious diseases. Infectious diseases, such as any fungal or bacterial infections, are contemplated for detection using the subject matter of the present disclosure. As used herein, the term "infection" refers to the invasion of the body tissues of a host organism by disease-causing organisms, their proliferation, and the host tissue's response to these organisms and the toxins they produce. Infections include, but are not limited to, hospital-acquired infections, surgical infections, and severe abdominal infections such as peritonitis, pancreatitis, gallbladder empyema, and pleural empyema, as well as bone infections such as osteomyelitis. Detection of sepsis, sepsis, and septic shock, infections resulting from or following the use of immunosuppressive drugs, cancer chemotherapy, radiation, contaminated intravenous fluids, hemorrhagic shock, ischemia, trauma, cancer, immunodeficiency, viral infection, and diabetes are also contemplated. Examples of microbial infections, such as bacterial and / or fungal infections, include, but are not limited to, infections caused by Mycobacterium tuberculosis, Escherichia coli, Klebsiella, Enterobacter, Proteus, Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus (including Staphylococcus aureus and coagulase-negative staphylococci), Enterococcus, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides, Acinetobacter, Helicobacter, and Candida. Infections caused by resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE), are also included. In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a chronic bacterial infection. In some embodiments, the bacterial infection is tuberculosis. In some embodiments, the infection is disseminated tuberculosis. In some embodiments, the infection may be hepatitis A, hepatitis B, hepatitis C, and / or human immunodeficiency virus.

[0063] In some embodiments, imaging methods are used to detect inflammation.Examples of inflammation-related disorders include, but are not limited to, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis media, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, lupus, including systemic lupus erythematosus, and vasculitis.In some embodiments, inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus.

[0064] PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells, and regulates activation or inhibition. Thus, imaging agents of the present disclosure that detect PD-L1 expression can be used to detect immune cells such as T cells, B cells, and myeloid cells. In some embodiments, imaging agents of the present disclosure detect immune cells in tumors. In some embodiments, imaging agents of the present disclosure detect the distribution of immune cells systemically in a subject. In some embodiments, imaging methods are used to detect immune cell responses in infectious cells. In some embodiments, imaging methods are used to detect immune cell responses in inflammatory cells.

[0065] In some embodiments, the imaging methods of the present disclosure detect and / or measure changes in PD-L1 expression, such as treatment-induced changes in PD-L1 expression. Such methods can be used to confirm the efficacy of a particular treatment method and / or to determine an effective therapeutic dose range.

[0066] III. Kits Containing Contrast Agents In some embodiments, as described above, the presently disclosed subject matter provides kits for detecting programmed death-ligand 1 (PD-L1), the kits comprising an imaging agent comprising a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, when present, links the peptide and the reporting moiety, and wherein, when the linker is not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide.

[0067] Typically, the kits of the presently disclosed subject matter include an imaging agent of the present disclosure and instructions for practicing at least one of the presently disclosed methods. The imaging agent is usually provided in the kit in an amount sufficient to detect PD-L1 at least once in at least one subject or patient. The kit may also include some or all of the other reagents and supplies necessary to practice at least one embodiment of the presently disclosed method.

[0068] In its simplest form, the kits of the presently disclosed subject matter include a container containing at least one imaging agent of the presently disclosed subject matter. In some embodiments, the kits include multiple containers, each of which may contain at least one imaging agent or other material useful for practicing one or more embodiments of the methods of the present disclosure.

[0069] The container can be any material suitable for containing the composition of the present disclosure or other substances useful for carrying out the method of the present disclosure. Thus, the container can be a vial or an ampule. It can be made of any suitable material, such as glass, plastic, metal, or paper or paper products. In embodiments, it is a glass or plastic ampule or vial that can be sealed with a stopper, a stopper and crimp seal, or a plastic or metal cap. The amount of contrast agent contained in the container can be selected by those skilled in the art without undue experimentation based on numerous parameters related to the subject matter of the present disclosure.

[0070] In embodiments, the container is typically provided as a component of a larger unit that includes packaging materials (hereinafter referred to as a kit for brevity). The kits of the present disclosure may include appropriate packaging and instructions and / or other information regarding the use of the composition. Typically, kits are manufactured from sturdy materials such as cardboard or plastic, and instructions and other information may be printed directly onto them. The kits may include multiple containers containing the compositions of the present invention. In such kits, each container may be the same size and contain the same amount of composition as each of the other containers, or different containers may be different sizes and / or contain different amounts of composition or compositions with different ingredients. Those skilled in the art will readily recognize that many different configurations of container sizes and contents are contemplated by the present invention, and therefore, not all permutations need be specifically listed herein.

[0071] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this herein-described subject matter belongs.

[0072] Following long-standing patent law convention, the terms "a," "an," and "the" when used in this application, including the claims, refer to "one or more." Thus, for example, a reference to a "subject" includes a plurality of subjects unless the context clearly indicates otherwise (e.g., a plurality of subjects, etc.).

[0073] Throughout this specification and claims, the terms "comprise," "comprise," and "including" are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term "containing" and its grammatical variations are intended to be open-ended, such that the recitation of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0074] For purposes of this specification and the appended claims, unless otherwise indicated, amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, amounts, properties, and other numerical values ​​used in this specification and claims are to be understood as being modified in all instances by the term "about," even if the term "about" does not explicitly appear in conjunction with that value, amount, or range. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are not, and need not be, exact, but may be approximate and / or larger or smaller, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those of ordinary skill in the art that depend upon the desired properties sought to be obtained by the subject matter of the present disclosure. For example, the term "about" when referring to a value can mean encompassing, in some embodiments, ±100%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% variation from the specified amount, where such variation is appropriate to practice the disclosed methods or employ the disclosed compositions.

[0075] Furthermore, the term "about," when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including every number within the range, and modify that range by extending the boundaries above and below the numerical values ​​set forth. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., whole integers, including fractions thereof subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 2, 3, 4, and 5, and fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.), and any range within that range. [Example]

[0076] The following examples are included to provide guidance to those skilled in the art for carrying out representative embodiments of the presently disclosed subject matter. In light of this disclosure and the general state of the art, those skilled in the art will recognize that the following examples are intended to be illustrative only, and that numerous changes, modifications, and substitutions can be made without departing from the scope of the presently disclosed subject matter. The following synthetic descriptions and specific examples are intended for illustrative purposes only and should not be construed as limiting in any way to prepare compounds of the present disclosure by other methods.

[0077] Example 1 Rapid tumor PD-L1 detection with PET using a highly specific peptide 1.1 Background: Increased PD-L1 in the tumor microenvironment (TME) leads to immunosuppression by inactivating active immune infiltrates through binding to the programmed cell death protein 1 (PD-1) receptor expressed by the immune infiltrate (Okazaki et al., 2007, and Topalian et al., 2015). PD-L1 expression on tumor cells and in the TME is considered a potential biomarker for patient stratification and therapeutic monitoring (Herbst et al., 2014). A supplemental diagnostic test based on PD-L1 IHC was recently approved by the US Food and Drug Administration, suggesting that PD-L1 may be a suitable target for in vivo imaging (Roach et al., 2016).

[0078] Currently, immunohistochemistry (IHC) detection is the most studied predictive biomarker for therapeutic monitoring of PD-L1 / PD-1-targeted therapies. However, this approach and the available FDA-approved diagnostic IHC tests for PD-L1 have significant limitations (Roach et al., 2016; Mansfield and Dong, 2016; and Phillips et al., 2015). These limitations include inconsistent definitions of antigen positivity, discrepancies in the detection antibodies, poor agreement between assays, and intratumor and intertumor heterogeneity that undermine accuracy and reliability, thus hindering therapeutic decision-making. Furthermore, tissue samples obtained by biopsy for testing are typically very limited and may require molecular profiling to identify targetable oncogenic mutations in other pathways (e.g., epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase, DNA repair genes) that confer sensitivity or resistance to existing therapies. Such precious samples often make it impractical to perform multiple PD-L1 assessments for reliable delineation of PD-L1 expression. Novel PET imaging agents that allow noninvasive assessment of PD-L1 expression levels, kinetics, and distribution, and do so within the standard clinical workflow of imaging within 60 minutes of administration, would overcome the shortcomings of available (IHC-based) methods for assessing PD-L1 expression status.

[0079] The dynamic nature of the tumor immune microenvironment provides a rationale for the development of PET tracers that enable rapid assessment of the TME. In this regard, low-molecular-weight peptide-based PET tracers are desirable candidates for clinical application due to their fast clearance and ease of synthesis (Reubi et al., 2008; Sun et al., 2016). Peptide-based PET tracers targeting somatostatin receptors and chemokine receptor 4 (CXCR4) produce high target-to-nontarget ratios in patients (Herrmann et al., 2016; Gourni et al., 2011).

[0080] Recently, peptides that specifically bind to PD-L1 have been reported (see International PCT Patent Application Publication No. WO2016039749, published March 17, 2016, by Miller et al., Macrocyclic Inhibitors of the PD-1 / PD-L1 and CD80 (B7-1) / PD-L1 Protein / Protein). Interactions; International PCT Patent Application Publication No. WO2016 / 100285, "Immunomodulators," published June 23, 2016; International PCT Patent Application Publication No. WO2016 / 100608, "Immunomodulators," published June 23, 2016; International PCT Patent Application Publication No. WO2016 / 126646, "Immunomodulators," published August 11, 2016, each of which is incorporated herein in its entirety; however, their potential for detecting PD-L1 expression in vivo has not been established. It was hypothesized that these PD-L1-binding peptides have the potential to rapidly and specifically detect PD-L1 expression in tumors. To test this hypothesis, a peptide, WL12, was selected from a reported peptide library that possesses a single primary amine and is most suitable for conjugation, and its binding mode to PD-L1 was evaluated. DOTAGA chelator 64 For radiolabeling with Cu, conjugate with WL12 [ 64 Cu]WL12 (Eisenwiener et al., 2000) to evaluate the binding affinity of peptide derivatives to PD-L1 and to compare the binding affinity of peptide derivatives to PD-L1 in cell lines with variable PD-L1 expression. 64 As a proof of concept, we evaluated the in vitro uptake of [Cu]WL12. 64 The ability of Cu]WL12 to detect PD-L1 expression by PET imaging in vivo was evaluated in NSG mice bearing Chinese hamster ovary (CHO) tumors with constitutive human PD-L1 expression (hPD-L1) and isogenic negative control tumors (CHO). 64 The tissue distribution and target specificity of [Cu]WL12 were confirmed by ex vivo biodistribution and blocking studies.

[0081] (1.2 Results and Discussion) 1.2.1: WL12 binds to PD-L1 in a manner similar to PD-1. To evaluate the binding mode of WL12 to PD-L1, we used the co-crystal structure of human PD-L1 bound to PD-1 (PDB ID: 4ZQK) (Zak et al., 2015) to dock WL12 in place of PD-1. Given the structural complexity of the macrocycle WL12, we first performed a conformational search and docked conformers into the PD-1 binding site on PD-L1 using Glide (Friesner et al., 2004; Halgren et al., 2004). WL12 forms a beta-sheet-like structure with two hydrogen bonds between the backbones of the two macrocyclic chains (Figure 1B). This conformation is supported by circular dichroism experiments (Figure 2). Superimposing the structure of PD-1 with bound WL12 reveals the similarity in the binding mode between these two. The two beta strands of PD-1, which form the binding interface with PD-L1, overlap with the pseudostrand of WL12 (Figure 1C). The L-leucine of WL12 inserts into the same small hydrophobic pocket as Ile134 of PD-1, and one of the two norleucine residues aligns with Ile126 of PD-1. In addition to these hydrophobic interactions, numerous hydrogen bonds exist between WL12 and PD-L1. The asparagine carboxamide on WL12 forms a hydrogen bond with Tyr123, the glycinamide forms a hydrogen bond with the backbone of Gly120, and the serine hydroxyl interacts with Gln66. The ornithine residue is exposed and does not participate in binding to PD-L1. While not wishing to be bound by any one particular theory, this suggests that conjugation of an appropriate label via amine coupling does not interfere with WL12 binding to PD-L1.

[0082] 1.2.2:[ 64 Cu]WL12 exhibits PD-L1-specific cellular uptake in vitro. 13 Ornithine (Orn) primary amine is utilized to conjugate DOTAGA, which is then used to generate non-radioactive C 2+An analogue (WL12-Cu) was prepared, 64 The resulting WL12D and corresponding WL12-Cu were purified by HPLC, characterized by mass spectrometry (Figures 2, 3, 4, 5, 6, and 7), and subjected to in vitro evaluation. The half-maximal inhibitory concentrations (IC) of WL12 and its derivatives that inhibit the interaction of PD-L1 with PD-1 were determined. 50 We optimized a previously described in vitro assay relying on fluorescence resonance energy transfer (FER) to assess the IC values ​​of WL12, WL12D, and WL12-Cu (Woodard et al., 2014). We obtained IC values ​​of 22, 23, and 2.9 nM for WL12, WL12D, and WL12-Cu, respectively. 50 Values ​​were observed (Figures 8A, 9, 10 and Table 1 below). These data 13 Orn side chain was modified with DOTAGA and Cu 2+ These results indicate that WL12 retains high binding affinity for PD-L1 even when chelated to PD-L1.

[0083] [Table 1]

[0084] To demonstrate PD-L1 specificity and cellular uptake, 64 Cu]WL12 was produced with high specific activity (1.9±0.1 mCi / μg) and radiochemical purity (>95%) (Figures 11 and 12). 64 hPD-L1 cells incubated with [Cu]WL12 for 1 hour showed >50% uptake of the incubated dose and a 43-fold increase in bound radioactivity compared to negative control CHO cells (Figure 8C). 64 Binding specificity was tested by incubating with [Cu]WL12 alone or in the presence of a blocking dose of 1 μM WL12. 64 A >95% reduction in Cu]WL12 was observed in the presence of the peptide, and [ 64 The binding of [Cu]WL12 was shown to be specific (Figure 8C). 64The efficacy of [Cu]WL12 was further tested in two triple-negative breast cancer (TNBC) cell lines, MDAMB231 and SUM149, which exhibit high and low PD-L1 expression, respectively (Figure 8B). The two-fold higher uptake of radioactivity in MDAMB231 cells compared to SUM149 cells indicated a [Cu]WL12 inhibitory effect on PD-L1. 64 The specificity of [Cu]WL12 was further confirmed (Figure 8C). Flow cytometry analysis of PD-L1 expression showed mean fluorescence intensity values ​​in the following order: hPD-L1 > MDAMB231 > SUM149 > CHO, which correlated with radioactivity incorporation (r=0.9977, Figures 13 and 14). Collectively, these results demonstrate that [ 64 We demonstrate that Cu]WL12 binds to cancer cells in vitro in a PD-L1 expression-dependent manner.

[0085] 1.2.3:[ 64 Cu]WL12 specifically accumulates in tumors with high PD-L1 expression. 64 To gain insight into the in vivo specificity and distribution of [Cu]WL12, PET-CT imaging studies were performed in mice bearing hPD-L1 and CHO tumors (n=4). PET imaging studies revealed that [Cu]WL12 in hPD-L1 tumors 64 Cu]WL12 demonstrated robust uptake of hPD-L1. Increased uptake in hPD-L1 tumors was observed as early as 10 minutes and persisted up to 24 hours post-injection (Figures 15A and 16), and PD-L1 expression was confirmed by IHC (Figure 15B). In addition to tumors, high uptake was also observed in the kidney and liver. To confirm the PET imaging observations, biodistribution studies were performed [ 64Cu]WL12 (n=3 and n=5, respectively). Given the rapid uptake observed in PD-L1-positive tumors, biodistribution at 1 and 2 hours was considered more informative for the development of 18F-labeled analogs. Consistent with imaging studies, hPD-L1 tumors demonstrated radioactivity uptake at 1 hour with a percent of injected dose / g (%ID / g) value of 14.9±0.8. In contrast, control CHO tumor uptake was 4.0±0.6%ID / g (Figure 17). Uptake in the kidney and liver was also relatively high, with uptake values ​​of 34.4±3.1 and 24.2±2.5%ID / g, respectively. The tumor-to-muscle and tumor-to-blood ratios for hPD-L1 tumors were 25.6±1.9 and 4.7±1.2, respectively, indicating a significant correlation between the uptake of 18F-labeled analogs and the tumor-to-muscle and blood ratios. 64 This was consistent with the ability of [Cu]WL12 to provide PD-L1-specific images with a high signal-to-noise ratio (Figures 15A and 15B).

[0086] Biodistribution studies performed after 2 hours showed a similar profile with a trend toward decreased radioactivity in the kidney, liver, and tumor (Figure 17). 64 [Cu]WL12 was co-injected with excess WL12 (50 μg, 2 mg / kg) and biodistribution studies were performed at 2 hours. A >75% decrease (P<0.0001) in %ID / g values ​​was observed in hPD-L1 tumors, with no significant difference observed in control CHO tumors. A decrease in uptake was also observed in the kidney. No significant differences in radioactivity uptake were observed in other tissues. Increased uptake in the liver, 64 A trend often observed with Cu-based contrast agents (Anderson et al., 2009) is the loss of Cu from the chelating agent. 2+This may be due to dissociation of PD-L1 and subsequent transchelation to plasma proteins such as albumin and ceruloplasmin (Smith-Jones et al., 1991; Wadas et al., 2007; and Boswell et al., 2004). The increased renal uptake also primarily suggests renal clearance of the peptide. Low uptake was observed in the spleen, thymus, and brown fat, tissues known to express PD-L1 and reported to show increased uptake of radiolabeled antibodies (Chatterjee et al., 2016; Hettich et al., 2016; and Josefsson et al., 2016). 64 These results suggest that [Cu]WL12 has very low or no affinity for mouse PD-L1. 64 Further supporting the specificity of [Cu]WL12, no significant differences in uptake were observed between the control and blocking dose groups in these tissues, except for the kidney. Imaging and biodistribution studies were performed jointly to demonstrate the specificity of [Cu]WL12. 64 We demonstrate that [Cu]WL12 rapidly and specifically binds to human PD-L1.

[0087] 1.2.4: CD Results. To assess the secondary structure of WL12 in aqueous and membrane-mimetic solutions, CD spectroscopy was performed in a combination of water, DPC, and SDS. As shown in Figure 2, Trp residues significantly influence the CD spectrum of the WL12 peptide in the 220-240 nm region. In detergent-free solutions, a minimum at approximately 220 nm and a positive shoulder at approximately 230 nm are observed. Upon addition of detergent, both bands are slightly red-shifted, with the latter exhibiting an increase in intensity. These bands are attributed to Trp-Trp coupling. Both Trp chromophores are in close proximity, and they behave as a single absorbing unit. As a result, their excited states interact, and the excited state of the dimer system is delocalized across both monomers. This phenomenon, called the exciton effect, causes the excited state to split into two components, one resulting from the in-phase coupling of the two monomer excitations and the other from the out-of-phase coupling (Grishina 1994, and Kelly 2000).

[0088] CD spectra of disordered peptides are typically characterized by a single band below 200 nm, whereas α-helices exhibit two negative bands at 208 and 222 nm and one positive band at 192 nm. β-sheet structures typically exhibit a negative band at 217 nm and a positive band at 195 nm. Therefore, the strong negative band at ~205 nm and the strong positive band at ~190 nm in the CD spectrum of the WL12 peptide may suggest a mixture of random coil and more ordered structures. Deconvolution of the CD spectrum indicates a high β-sheet content (~40%) under all measurement conditions. Nevertheless, the strong contribution of the Trp chromophore to the far-UV CD spectrum of WL12 affects the accuracy of quantitative analysis of secondary structure content, and the results should be interpreted with caution.

[0089] 1.3 Summary: In summary, rapid tumor PD-L1 detection and PD-L1 selectivity were demonstrated using highly specific PD-L1 binding peptides [ 64 This has been demonstrated in vitro and in vivo by PET using [Cu]WL12. 64 The pharmacokinetics and biodistribution of [Cu]WL12 suggest that PD-L1 detection is feasible to fit into standard clinical workflows, imaging patients within 60 minutes of radiotracer administration. Rapid, noninvasive detection of PD-L1 expression across all malignant lesions offers an unprecedented opportunity to stratify patients for immunomodulatory therapy.

[0090] 1.4 Materials and Methods 1.4.1 Materials: The PD-L1 binding peptide, WL12, was custom synthesized by CPC Scientific (Sunnyvale, CA) with a purity of >95%. Unless otherwise specified, all other chemicals were purchased from Sigma-Aldrich or Fisher Scientific. 2,2',2'-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA anhydride) and [ 64 CuCl2 was purchased from CheMatech Macrocycle Design Technologies (catalog no. C109; Dijon, France) and the University of Wisconsin, respectively. All cell culture-related reagents were purchased from Invitrogen unless otherwise specified. Polyclonal anti-human IgG-Eu3+ cryptate (catalog no. 61HFCKLA) and XL665-conjugated mouse monoclonal anti-6 histidine antibody (catalog no. 61HISXLA) were purchased from Cisbio Assays (Bedford, MA). Recombinant human PD-1 Fc chimeric protein (catalog no. 1086-PD-050) and recombinant human PD-L1 (B7-H1)-His-tag protein (catalog no. 9049-B7) were obtained from R&D systems (Minneapolis, MN).

[0091] 1.4.2 Docking Study: To perform docking of WL12 to PD-L1, the crystal structure of human PD-1 bound to PD-L1 (PDB ID: 4ZQK) was used as a template. The model was first prepared using Maestro's Protein Preparation Wizard (Schrodinger Release 2016-2: Maestro, version 10.6, Schrodinger, LLC, New York, NY, 2016) (Sastry et al., 2013). This involved assigning bond orders and formal charges, adding hydrogen atoms, and adding missing side chains. The hydrogen bond network within the protein was optimized (including reorienting thiol and hydroxyl groups, sampling Asn, Gln, and His side chains, and predicting the protonation states of His, Asp, and Glu), followed by a simple minimization. The PD-1 structure was then removed. A conformational search was performed on the WL12 structure using Prime Conformational Search (Schrodinger Release 2016-2: Prime, version 4.4, Schrodinger, LLC, New York, NY, 2016). The 100 lowest-energy conformers were selected for the docking experiment. Docking was performed with Glide (Schrodinger Release 2016-2: Glide, version 7.1, Schrodinger, LLC, New York, NY, 2016) using default settings and the input ring conformation (Friesner et al., 2004; Halgren et al., 2004). The software used for these calculations was SBGrid (Morin et al., 2013).

[0092] 1.4.3 Circular dichroism (CD) measurements: CD spectra of the peptide in detergent-free aqueous solution, in aqueous micellar solutions of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulfate (SDS), and in mixed DPC:SDS micelles at a molar ratio of 5:1 were obtained using a Jasco J-815 spectropolarimeter (Jasco, Easton, MD). All measurements were performed at 25 °C using a 0.15 mg / mL peptide solution. Experiments were performed over the range of 185–260 nm and in triplicate to increase the signal-to-noise ratio. Final spectra were corrected by background subtraction and the mean residue molar ellipticity, MRME (degrees × cm), was calculated. 2 ×dmol -1 ) versus wavelength λ (nm). Secondary structure content was calculated from the spectra using the CONTIN method (Sreerama et al., 2000).

[0093] 1.4.4: Synthesis of WL12-DOTAGA (WL12D): 3 mg of peptide (1.5 μmol) was dissolved in 0.5 mL of DMF and mixed with 3.7 mg of anhydrous DOTAGA (7.51 μmol in 0.5 mL of DMF) and 20 μL of diisopropylethylamine (DIPEA). The reaction mixture was stirred at room temperature for 2 h, and the product was purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system (Varian ProStar) with an Agilent Technology 1260 Infinity Photodiode Array Detector (Agilent Technologies, Wilmington, DE) on a semi-preparative C-18 Luna column (5 mm, 10 × 250 mm, Phenomenex, Torrance, CA) and a gradient elution starting with 98% HO (0.1% TFA) and 2% MeOH (0.1% TFA) at a flow rate of 4 mL / min, reaching 100% MeOH in 60 min. The desired WL12D was collected at 44.5 min, evaporated, dissolved in deionized water, and lyophilized to give 3.1 mg (1.3 μmol) of product as a white powder (yield: 82.9%, Figure 2). The resulting conjugate was solubilized in 50% (v / v) HO-MeOH containing 0.1% formic acid and analyzed by electrospray ionization mass spectrometry (ESI MS, Esquire 3000 Plus spectrometer, Bruker Daltonics, Billerica, MA) (Figure 4). Theoretical chemical formula: CHNOS. Observed ESI-MS m / z: 2340.9 (M+1). +1 , 1171.1-(M+2) +2 / 2 and 781.1-(M+2) +3 / 3. (Predicted value: 2340.65)

[0094] 1.4.5:WL12-Cu 2+Complex preparation: 1.5 mg of WL12D (0.64 μmol) was dissolved in 200 μL of sodium acetate (0.1 M, adjusted to pH 4.5 with glacial acetic acid), and 55 μL of 0.02 M aqueous CuCl2 solution (1.1 μmol) was added. The resulting reaction mixture was incubated at 65 °C for 30 min, purified by RP-HPLC as described for WL12D (Figure 5), lyophilized, and the resulting pale blue powder was analyzed by ESI MS (Figure 6). WL12-Cu was then used as a radiolabeled standard and as a standard for PD-L1 and PD-1 competitive binding assays. 2+ The RP-HPLC conditions were optimized using the complex (Figure 7). Theoretical formula: C110H156N26O29S. Observed ESI-MS m / z: 2402.6 -(M+1) +1 , 1201.9 -(M+2) +2 / 2 (predicted value: 2402.18)

[0095] 1.4.6: PD-L1 and PD-1 Binding Inhibition Assay: A competitive inhibition assay of PD-L1 binding to PD-1 was optimized from a previously described fluorescence resonance energy transfer (FRET)-based assay in discussions with Cisbio (Woodard et al., 2014). All binding / inhibition assays were performed in 21 μL of FRET assay buffer (dPBS, bovine serum albumin (0.1%, w / v), Tween-20 (0.05% v / v), and sodium fluoride (400 mM)). Assay conditions were first optimized for PD-1 and PD-L1 concentrations. PD-1-Ig at final concentrations of 10 nM, 20 nM, and 40 nM was incubated with PD-L1-His-tag at final concentrations ranging from 0.65 to 320 nM (each concentration in triplicate) for 15 min, followed by anti-human IgG-Eu. 3+Ten microliters of FRET buffer containing cryptate (IgG-Eu, final concentration 2 nM) and anti-6HIS-XL665 monoclonal antibody (anti-6HIS-XL665, final concentration 40 nM) was added. After 1 hour of incubation at room temperature, 1 μL of NaF assay buffer was added (final concentration, 400 mM), and the plate was read using a Perkin Elmer Victor3 1420 multilabel counter (Perkin Elmer, Waltham, MA).

[0096] For competitive inhibition assays, inhibitors (WL12, WL12D and WL12-Cu 2+ , range: 1 pM–1 mM) was preincubated with PD-L1-His-tag (final 80 nM) in 10 μL of assay buffer for 15 minutes. Subsequently, 5 μL of assay buffer containing PD-1-Ig (final concentration 20 nM) was added and incubated for 15 minutes. Next, 5 μL of assay buffer containing IgG-Eu (final concentration 2 nM) and anti-6HIS-XL665 (final concentration 40 nM) was added. After 1 hour of incubation at room temperature, 1 μL of NaF was added (final concentration 400 mM), and the plate was read on a Perkin Elmer Victor3 1420 multilabel counter. IC50 and Ki values ​​were calculated by fitting the data to a sigmoidal dose-response curve and the Cheng-Prusoff equation, yielding a KD of 70 nM for PD-L1 at a concentration of 80 nM. All experiments were performed in triplicate and repeated three times.

[0097] 1.4.7.[ 64 Cu]WL12 generation: Purchased from University of Wisconsin 64 CuCl2 was evaporated to a small volume and titrated with 0.1 M sodium acetate solution. 64 For radiolabeling, approximately 10 μg of WL12D peptide conjugate (4.27 nmol) in 100 μL of sodium acetate was converted to Cu(OAc). 64The mixture was mixed with Cu(OAc)2 and incubated at 65°C for 30 minutes. The resulting radioactive tracer was purified on a C-18 (Luna, 5 μm, 10 x 250 mm; Phenomenex) semi-preparative column using a Varian ProStar system (Model 105S; Bioscan, Poway, CA) equipped with a radioactive single-channel radiation detector and a Varian ProStar UV absorbance detector set at 280 nm. A gradient elution was applied, starting with 98% HO (0.1% TFA) and 2% MeOH (0.1% TFA) and reaching 90% MeOH over 70 minutes at a flow rate of 5 mL / min. 64 Cu]WL12 was collected at ~56.2 min (retention time of unlabeled peptide: 53.6 min), evaporated, and diluted with saline containing 5% DMSO and two drops of Tween 20 for in vitro and in vivo evaluation. 64 Cu]WL12 was obtained in a yield of 52.09 ± 6.3% with a specific activity of 1.9 ± 0.11 mCi / μg.

[0098] 1.4.7. Cell Lines: The Chinese hamster ovary cell line CHO-K1 (hereafter referred to as CHO) and the triple-negative breast cancer (TNBC) cell line MDAMB231 were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and passaged for less than three months, after which new cultures were started from vials of frozen cells. The SUM149 cell line was a gift from Dr. Stephen P. Ethier of the Medical University of South Carolina and authenticated by STR profiling at the Johns Hopkins Genetic Resource Facility. SUM149 cells were maintained in Ham's F-12 medium containing 5% FBS, 1% P / S, 5 μg / mL insulin, and 0.5 μg / mL hydrocortisone. All other cell lines were cultured in ATCC-recommended media in an incubator at 37°C in an atmosphere containing 5% CO2. A CHO cell line stably expressing human PD-L1 (hereafter referred to as hPD-L1) was generated in our laboratory ( Chatterjee et al., 2016 ) and maintained in F-12K medium containing 10% FBS, 1% P / S, and 2% mg / mL G418.

[0099] 1.4.8. Flow Cytometry: Cells in suspension were harvested by centrifugation, and adherent cells were detached using enzyme-free, PBS-based cell dissociation buffer (Thermo Fisher Scientific, Waltham, MA). Harvested cells were washed twice with flow cytometry buffer (1x PBS containing 2mM EDTA and 0.5% FBS). Cells were stained with phycoerythrin-conjugated anti-human PD-L1 antibody (BD-MIH-PE, clone number MIH1, catalog number 557924, Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer's protocol and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). At least 20,000 events were recorded.

[0100] 1.4.9. In vitro binding: hPD-L1 to CHO, MDAMB231, and SUM149 cells 64 In vitro binding of [Cu]WL12 was measured using 1 μCi of radiotracer at 1 × 10 6 The IgG was determined by incubating with cells at 37°C for 1 hour. After incubation, cells were washed three times with cold PBS before being counted in an automated gamma counter (1282 Compugmama CS, Pharmacia / LKB Nuclear, Gaithersburg, MD). 64 To demonstrate PD-L1-specific binding of [Cu]WL12, PD-L1 blocking was performed with 1 μM WL12 peptide or the humanized anti-PD-L1 antibody atezolizumab. Mean fluorescence intensity values ​​were correlated with the percent uptake of the incubated dose (%ID). All cellular uptake studies were performed in triplicate for each cell line and repeated three times.

[0101] 1.4.10. Animal Model: Animal experiments were performed in accordance with protocols approved by the JHU Animal Care and Use Committee (ACUC). Six- to eight-week-old female non-obese diabetic severe combined immunodeficiency gamma (NSG) mice were obtained from the JHU Immune Compromised Americal Core. Mice were inoculated with 10 x 10 IgG serogroups on both sides of the upper abdomen. 6 CHO-PDL1 and CHO cells were subcutaneously implanted. The tumors were 200-300 mm 3 When a volume of 100 μg / ml was reached, the mice were used for imaging or biodistribution experiments.

[0102] 1.4.11. PET-CT Imaging of Mouse Xenografts: Mice were injected with 150 μCi of [ 64 Mice were intravenously injected with [Cu]WL12 (n=3) and anesthetized under 3% isoflurane before being placed in the scanner. Mice were maintained at a 1% isofluorane level during imaging. PET images were acquired on an ARGUS small animal PET / CT scanner (Sedecal, Madrid, Spain) for 10 min / bed in two bed positions. A CT scan (512 projections) was performed at the end of each PET scan for anatomical coregistration. PET data were reconstructed using a two-dimensional ordered subsets expectation maximization algorithm (2D-OSEM) and corrected for dead time and radioactive decay. %ID values ​​per cc were calculated based on calibration coefficients obtained from known radioactivity. Final data visualization and image generation were achieved using Amira® (FEI, Hillsboro, OR).

[0103] 1.4.12. Ex vivo biodistribution: hPD-L1 and CHO tumor-bearing mice with high and low PD-L1 expression, respectively (n=5) were administered 40 μCi of [ 64 Cu]WL12 was injected intravenously. 64At 1 and 2 hours after [Cu]WL12 injection, blood, tumor, and selected tissues were collected, weighed, and counted in an automatic gamma counter (Perkin Elmer-2480 Automatic Gamma Counter-Wizard2 3' Wallac). For blocking studies, mice were co-injected with 2 mg / kg (50 μg) of unmodified peptide together with the radiotracer. Percentage of injected dose per gram of tissue (%ID / g) values ​​were calculated using signal decay correction and external [ 64 Calculations were based on normalization to a [Cu] standard. Biodistribution data shown are means ± standard error of the mean (SEM).

[0104] 1.4.13. Data Analysis: Statistical analysis was performed using unpaired two-tailed t-tests using Prism 6 software (GraphPad Software, La Jolla, CA). A P value of <0.05 was considered significant, and comparisons were made with cell lines or tumors with low PD-L1 expression. Flow cytometry data were analyzed using FlowJo software (Tree Star, Ashland, OR). IC 50 and Ki values ​​were calculated using Prism 6 software (GraphPad).

[0105] Example 2 (PD-L1-directed PET to PD-L1-targeted drug development) 2.1 Overview: Cancer immunotherapy (CIT) has improved patient survival by producing durable responses in a variety of malignancies. However, nearly 70% of patients treated with immune checkpoint-targeted therapy fail to respond to monotherapy (Lipson et al., 2015; Topalian et al., 2015). There is an unmet need to identify determinants of response to precision immunotherapy. Checkpoint combination therapy extends survival but often at the expense of increased immune-related adverse events (irAEs), suggesting that increased knowledge of combination strategies is necessary to mitigate toxicity (Marrone et al., 2016). There is a great need for focused research to identify new biomarkers for immune checkpoint therapy and their combinations that increase its breadth and durability and reduce irAEs. Therefore, one aspect of the subject matter of this disclosure is to develop strategies for using PD-L1-based PET imaging in the development and evaluation of PD-L1-targeted therapeutics. Unlike current strategies that rely on plasma or tissue (biopsy)-based biomarkers, which are invasive and impractical in advanced-stage patients, the present invention establishes the dose-occupancy relationship of PD-L1-targeted therapeutics (antibodies, peptides, small molecules) in relevant in vivo tumor models using PD-L1 PET imaging.

[0106] 2.1.1. Advances Enabled by PET-Based Quantification of PD-L1 Dynamics: It has recently been discovered that the accumulation of the PD-L1-targeted therapeutic AtzMab and its mouse chimera (PRO) in NSCLC, TNBC, and colon tumors is not entirely dependent on PD-L1 expression, as H2444 NSCLC xenografts with high PD-L1 expression accumulated significantly less radiolabeled AtzMab than those seen in breast cancer xenografts with low PD-L1 expression, as detected by IHC and flow cytometry (Chatterjee et al., 2016). Similarly, in a syngeneic mouse tumor model, systemically injected radiolabeled PRO was primarily associated with the tumor vasculature and showed little or no diffusion into the tumor parenchyma (Deng et al., 2016). Such findings may be due to pathophysiological features, including elevated interstitial pressure within tumors (Baxter et al., 1989; Baxter et al., 1990), which prevents the accumulation of therapeutic agents within tumors, which is an important factor in therapeutic resistance (Goel et al., 2011). Such effects may also potentially hinder the access of PD-L1-targeted therapeutic agents, which act primarily on tumor cells and tumor immune infiltrates. Therefore, WL12 / [ 18 Due to their very small molecular size, peptides such as [F]WL12 or similar radiolabeled peptides can penetrate tumor tissue and reach target cells more effectively and efficiently than antibodies. Therefore, by using appropriate analysis and correction, [F]WL12 can be used to detect and treat tumors. 18 [F]WL12 measurements or measurements made using similar radiolabeled peptides can help identify / optimize the therapeutic mAb dose required to target tumor cells to achieve the desired occupancy within the tumor tissue.

[0107] Therefore, PD-L1-directed PET has been applied to PD-L1-targeted drug development. To evaluate its potential value, innovative strategies [ 64Using [Cu]WL12, we evaluated and compared the tumor PD-L1 engagement characteristics of the therapeutic PD-L1 antibody atezolizumab (AtzMab) in terms of dose versus mAb localization in tumors as seen by PET. The preclinical findings disclosed herein may have clinically actionable implications. In patients, similar PD-L1 PET-based imaging measurements may be used to guide therapeutic dose intensification and improve treatment efficacy (Yang et al., 2013; Oude Munnink et al., 2016). Furthermore, such PD-L1 PET measurements may guide the future development of novel PD-L1-targeted therapeutics by enabling quantification of their potential target engagement at the tumor site.

[0108] 2.1.2. Innovations in the Use of PD-L1 PET in Drug Development and Evaluation: The disclosed innovative PD-L1 peptide-based PET imaging strategy enables assessment of the target engagement efficacy (i.e., occupancy and residence time) of current and future anti-PD-L1 therapeutics in tumors where they are most relevant. Dynamic PD-L1 density / turnover and the degree of PD-L1-expressing tumor burden, which influence serum mAb concentrations, along with the completeness of tumor perfusion and resulting intratumoral mAb accumulation, significantly impact therapeutic efficacy. While radiolabeled antibodies have previously been used to define required mAb dose levels and calculate target surface molecule occupancy (Deng et al., 2016), a key limitation of that approach is that it can only predict PD-L1 occupancy at the tumor site of action. The disclosed approach effectively addresses this issue and quantifies PD-L1 occupancy at the tumor site. Therefore, in addition to considering the contribution of key tumor physiological parameters to the effective mAb dose and cumulative dose achieved, we hope that our novel PET tracer-based measurements may improve the current understanding of why some patients with PD-L1-positive tumors fail to respond to CIT and may guide dose-escalation strategies to reach the desired tumor occupancy level.

[0109] 2.1.3. Evaluate the utility of PD-L1-PET in the development and evaluation of PD-L1-targeted therapeutics: (2.1.3.1 Rationale) Therapeutic antibodies targeting PD-L1 and PD-1 have shown excellent efficacy in a small subset of patients with PD-L1-positive tumors. At currently used doses, responder and non-responder populations show approximately 65% ​​PD-L1 occupancy in PBMCs, but the relationship between PD-L1 occupancy in PBMCs and occupancy in tumors is dynamic and poorly understood (Brahmer et al., 2012). Studies in tumor models have also found that PD-L1 antibodies are restricted to the tumor vasculature in some tumors (Deng et al., 2016). Preliminary results using radiolabeled AtzMab recapitulated these findings in NSCLC xenografts (Chatterjee et al., 2016). Collectively, these findings suggest that improved understanding of tumor PD-L1 occupancy and its dose-dependence, as well as the residence time of anti-PD-L1 antibodies in tumors, are necessary for better-informed PD-L1-directed therapy. Without wishing to be bound by any one particular theory, it is believed that PD-L1 PET provides a valuable tool for evaluating such PK measurements of anti-PD-L1 antibodies (or peptides and small molecules) with respect to target binding and residence time. It is also believed that PET-informed dosing leads to changes in the immune profile within the tumor that can be quantified by PD-L1 PET and correlated with treatment-induced changes in tumor PD-L1 expression and immune cell infiltration.

[0110] 2.1.3.2 Representative Data: Radiolabeled versions of available anti-PD-L1 antibodies and PD-1 derivatives have been used to noninvasively detect PD-L1 expression (Chatterjee et al., 2016; Deng et al., 2016; Hettich et al., 2016; Josefsson et al., 2016; Lesniak et al., 2016; Heskamp et al., 2015; Maute et al., 2015). To that end, the therapeutic antibody AtzMab was selected for its human and mouse cross-reactivity, and its specificity for PD-L1 detection was demonstrated by PET, SPECT, and optical imaging in human TNBC and NSCLC xenografts in immunocompromised mice and the 4T1 syngeneic breast tumor model (Chatterjee et al., 2016; Lesniak et al., 2016) (Figure 20A, Figure 20B, Figure 20C, and Figure 20D). AtzMab binds to both human and mouse PD-L1 with high affinity, with dissociation constants (Kd) of 0.43 nM and 0.13 nM, respectively (Irving et al., 2012; Powles et al., 2014). AtzMab is under clinical evaluation for the treatment of advanced or metastatic bladder cancer, (Powles et al., 2014) melanoma, (Hamid et al., 2013) NSCLC, (Spigel et al., 2013) RCC, (Cho et al., 2013) TNBC, and several other cancers.

[0111] Accumulation of radiolabeled AtzMab in tumors was found to be PD-L1 specific in both cancer types (NSCLC and TNBC) (Chatterjee et al., 2016; Lesniak et al., 2016). 111IWe also found that AtzMab intratumoral accumulation was not entirely dependent on PD-L1 expression, suggesting that interstitial fluid pressure, tumor convection, and spatial variations in extravasation are some of the contributing factors—a problem frequently observed with antibodies (Baxter et al., 1989). MDAMB231 TNBC xenografts showed higher tissue accumulation (percentage of injected dose per gram; %ID / g) than subcutaneous and orthotopic H2444 NSCLC tumors, which showed higher PD-L1 expression by both flow cytometry and IHC analysis (Chatterjee et al., 2016). By utilizing the specificity and flexibility of our novel peptide-based PD-L1 PET tracer, we analyzed the kinetics of AtzMab accumulation in PD-L1-expressing tumors in vivo, accounting for the numerous factors that influence antibody distribution within tumors—a distinct approach that can be applied to various PD-L1-targeting antibodies.

[0112] 2.1.3.3 Accumulation of PD-L1 Therapeutic Antibodies in Tumors by PD-L1 PET: While evaluating the specificity of WL12 for PD-L1, it was discovered that WL12 competes with AtzMab for the same binding site on PD-L1. This provides a novel and previously unanticipated means to evaluate AtzMab therapy at the tumor site where it is needed, using PD-L1-directed PET. Improved understanding of the distribution of PD-L1 antibodies in tumors may impact clinical antibody dosing and therapeutic monitoring. Therefore, assessing AtzMab binding to PD-L1 in tumors [ 64 The ability of [Cu]WL12-PET was tested. 64 Accumulation of radioactivity in hPD-L1 tumors was reduced by 80% in mice injected with AtzMab (20 mg / kg), as quantified by [Cu]WL12-PET and biodistribution studies (Figures 21A, 21B, and 21C). Radioactivity uptake in other tissues except the kidney was reduced, but no significant differences were observed. 64Cu] WL12 binding was shown to be specific for human PD-L1 (Lesniak et al., 2016). In vitro binding studies showed that unlabeled WL12 inhibited the binding of Cy5-conjugated AtzMab to PD-L1 in a concentration-dependent manner, with IC 50 was 37.8 nM, and both ligands compete for PD-L1 binding (Figure 21D), but AtzMab 64 We confirmed that [Cu] is more potent than WL12 in inhibiting WL12 binding, which should enable detection of unoccupied PD-L1 levels in tumors upon AtzMab administration. Collectively, these results demonstrate that the binding sites of WL12 and AtzMab overlap, providing a useful tool for assessing AtzMab target engagement and residence time (target engagement efficacy) in PD-L1-expressing tumors. 64 This demonstrates the potential utility of [Cu]WL12-PET. The applicability of this approach was extended to cancer cell lines with naturally elevated PD-L1 expression. In a triple breast cancer xenograft study, we detected AtzMab accumulation in the highly PD-L1-expressing MDAMB231 xenograft. 64 Cu]WL12 was observed (Figure 22). A significant decrease in PD-L1 PET imaging agent uptake in mice receiving the 20 mg / Kg AtzMab dose was also observed.

[0113] Similar applications are anticipated for this antibody, similar to other PD-L1-targeting therapeutic antibodies such as avelumab (AvMab). AvMab is a human IgG1 antibody currently undergoing multiple Phase III clinical trials in several cancers, including NSCLC (NCT02395172), advanced RCC, and gastric cancer. Analysis of the crystal structure of PD-L1 complexed with AvMab revealed that AvMab interacts with several of the same amino acids on PD-L1 (R113, D61, and E58) as WL12 (Liu et al., 2016), demonstrating the potentially advantageous utility of a WL12-based tracer for assessing in vivo target engagement by AvMab and potentially other PD-L1-directed therapeutic mAbs. These studies validate the potential of PD-L1-PET to evaluate ongoing PD-L1 mAb therapy for its target engagement potential.

[0114] Example 3 (Non-invasive quantification of PD-L1 engagement by theranostic antibodies) 3.1 Overview: Antibody therapeutics targeting programmed death-ligand-1 (PD-L1) have been employed in nearly one-quarter of clinical trials involving immune checkpoint inhibitors. The relationship between total PD-L1 levels, their occupancy by PD-L1 therapeutics, and the extent and duration of target engagement within tumors to ensure optimal immune responses remains unknown. PD-L1 occupancy within tumors may be influenced by dynamic changes in PD-L1 expression, as well as tumor intrinsic and extrinsic parameters that alter plasma and tumor antibody concentrations. However, such important variations are not captured by peripheral pharmacokinetic and pharmacodynamic assessments. To address gaps in the dose-drug exposure relationship of PD-L1 therapeutics, we investigated radiolabeled PD-L1-binding peptides, which enable quantification of dynamic changes in PD-L1 expression. Structural analysis demonstrated overlap in the interactions of peptides and therapeutic monoclonal antibodies (mAbs) with PD-L1, enabling the occupancy of therapeutic mAbs in tumors to be measured using positron emission tomography (PET). In multiple xenograft models, PET imaging and biodistribution studies demonstrated that variable PD-L1 expression and its saturation by therapeutic PD-L1 antibodies could be quantified. Furthermore, we measured tumor PD-L1 occupancy with three different antibodies and quantified the effects of dose and time on tumor PD-L1 occupancy. Peptide-based PD-L1 PET is a promising tool for optimizing dose and treatment regimens with the goal of reducing immune-related adverse events.

[0115] More specifically, the subject matter of this disclosure addresses the need to characterize PD-L1 expression levels and PD-L1 mAb target engagement in tumors in vivo using quantitative positron emission tomography (PET) imaging, which is useful for repeated measurements of target expression in tumors (Wilman et al., 2008) and drug development and evaluation, but is rarely used for receptor occupancy studies in oncology (Rathkopf et al., 2013), and has not been implemented for pharmacokinetic and pharmacodynamic evaluation of PD-L1 or PD-1 mAbs in particular (Peterson et al., 2008; Linden et al., 2006).

[0116] It binds to human PD-L1 with high affinity and specificity, producing high-contrast images within 120 minutes of radiotracer administration. 64 A small peptide radiolabeled with Cu, [ 64 Cu]WL12 was recently developed (Chatterjee et al., 2017). This example demonstrates the use of [ 64 We describe [Cu]WL12-PET to quantify dynamic changes in PD-L1 expression in experimental models of lung and breast cancer. We evaluate PD-L1 engagement by three different FDA-approved mAbs: atezolizumab, avelumab, and durvalumab (DurMab). 64 We evaluated the ability of [Cu]WL12 PET to noninvasively assess the relationship of PD-L1 mAb dose to the extent and duration of PD-L1 engagement in tumors.

[0117] 3.2 Background: Cancer immunotherapy (CIT) has demonstrated durable responses against a variety of malignancies. One of the preferred CIT targets is the checkpoint protein programmed death-ligand 1 (PD-L1). PD-L1 is expressed by many tumors as a means of evading tumor-infiltrating cytotoxic T cells (Topalian et al., 2016) and causes immunosuppression through direct binding to the PD-1 receptor (Okazaki et al., 2007; Topalian et al., 2015). Multiple PD-L1-targeting monoclonal antibody therapeutics (mAbs) that inhibit the PD-L1:PD-1 interaction are in clinical trials, and nearly 30% of patients receiving these treatments demonstrate durable responses (Topalian et al., 2015; Lipson et al., 2015). However, despite these successes, there is an incomplete understanding of the biological mechanisms contributing to aberrant response patterns, such as delayed or mixed tumor regressions, which pose clinical challenges and limit clinicians' ability to advance checkpoint therapy.

[0118] The therapeutic effects of anti-PD-L1 mAbs are thought to occur primarily within the tumor microenvironment (Toparian et al., 2015). However, pharmacodynamic (PD) data are limited; they do not reflect target engagement at the site of action (tumor), have been reported in only a limited number of studies, and have been obtained using peripheral blood mononuclear cells (PBMCs). For the PD-L1 antibody BMS-936559, uniform target occupancy rates of 64–70% have been reported for doses ranging from 0.1 to 10 mg / kg (Brahmer et al., 2012). Much remains unknown about the location of PD-L1 mAbs at their most relevant site, the tumor, and the relevance of administration to the degree and duration of target engagement to ensure an optimal immune response.

[0119] The most studied predictive biomarker for therapeutic monitoring of PD-L1 / PD-1-targeted therapy is PD-L1 immunohistochemistry (IHC) (Gibney et al., 2016). However, the method has significant limitations because it requires biopsy specimens, which are of limited availability, and may not accurately reflect the temporally dynamic immune-tumor microenvironment (TME) and intratumor and intertumor heterogeneity of PD-L1 expression (Mansfield et al., 2016; McLaughlin et al., 2016). There is an unmet need for noninvasive assessment of PD-L1 expression levels, kinetics, and pharmacokinetics of PD-L1 therapeutics in primary and metastatic tumors, and to do so within the standard clinical workflow of imaging.

[0120] (3.3 results) 3.3.1: Structural analysis and in vitro validation of PD-L1 interaction with WL12 and PD-L1 mAb. WL12 is a 14-amino acid peptide that inhibits PD-L1:PD-1 interaction with high affinity (IC50: 20 nM) (Chatterjee et al., 2017). Initial molecular modeling analysis suggested overlapping interaction surfaces of PD-L1:WL12 and PD-L1:PD-1, with four amino acids (Y56, E58, D61, and A113) of PD-L1 contributing to the key molecular interaction (Chatterjee et al., 2017). The buried surface (2,106 Å) of PD-L1 in complex with the therapeutic antibody atezolizumab (AtzMab) was 2 ) is larger than that of PD-1 (1,970 Å 2) (Lee et al., 2017). Without wishing to be bound by any one particular theory, it is believed that the WL12 interaction surface on PD-L1 also overlaps with that of clinically available therapeutic mAbs because they are similarly designed to inhibit the PD-L1:PD-1 interaction. To test this, the predicted binding conformation of WL12 was compared with that of PD-L1 mAbs. Overlap of AA contacts in all mAbs, as well as PD-1 and WL12, reveals a common binding domain consisting of PD-L1 residues Y56, E58, A113, M115, and Y123. As revealed in visualizations of the PD-L1 molecular surface (Figures 33A and 34A), the overlapping region (cyan) forms a deep pocket and acts as an anchor point for all interaction points. In terms of surface area, AtzMab (red) interacts more with the PD-L1 surface with loops from the antibodies making molecular contacts with residues on all sides of the common binding core that overlap with interaction surfaces from PD-1 (purple), WL12 (green), avelumab (AveMab, orange), and durvalumab (DurMab, blue).

[0121] To confirm the structural analysis described above, Cy5-labeled AtzMab, AveMab, and DurMab were prepared by conjugation of the antibodies to commercially available Cy5 fluorescent N-hydroxysuccinimide esters, followed by competitive inhibition assays with WL12 in CHO cells constitutively expressing PD-L1 (Cho-hPD-L1) and MDAMB231 breast cancer cells naturally expressing PD-L1 (Chatterjee et al., 2016). WL12 dose-dependent inhibition of Cy5-PD-L1 mAb binding to PD-L1 was observed at inhibitory concentrations between 2 and 5 nM (Figure 3B). HCC827 and H226 non-small cell lung cancer (NSCLC) cells, each of which naturally expresses PD-L1, were also tested. They were incubated with fluorescent forms of AtzMab, AveMab, and DurMab in the presence of 5 nM WL12. Flow cytometry showed a significant decrease in binding fluorescence (P<0.001), further demonstrating the ability of WL12 to disrupt antibody-PD-L1 interactions (Figures 34C and 34D). Further confirmation of the specificity of the WL12:PD-L1 interaction was obtained when no change in binding fluorescence was observed using the CXCR4-specific antibody, MDX1338. PD-L1-positive (HCC827, H226, MDAMB231, and hPD-L1) and PD-L1-negative (Sum149 and CHO) cells were treated with 64Cu-radiolabeled WL12 analogs ([ 64 The WL12 analog was previously demonstrated to bind PD-L1 with high affinity (IC50<20nM) and selectivity in vitro and in vivo in hPD-L1 / CHO cells, but has not been validated in human cancer cell lines with variable expression (Chatterjee et al., 2017). The WL12 analog was found to bind PD-L1 with high affinity (IC50<20nM) and selectivity in PD-L1-positive cells compared to PD-L1-negative cells. 64 Furthermore, as a further check on PD-L1 binding specificity, a high expression-dependent uptake of [Cu]WL12 was observed in all PD-L1-positive cells when treated with 60 nM mAbs compared to PBS-treated controls (P<0.0001). 64 A significant blockade (P<0.0001) of [Cu]WL12 uptake was observed (FIG. 33C). 64We demonstrate that [Cu]WL12 can be used to detect free PD-L1 levels in tumors and monitor PD-L1 engagement by PD-L1 mAbs.

[0122] 3.3.2 Quantification of Tumor PD-L1 Engagement by AtzMab. To noninvasively assess PD-L1 engagement by therapeutic mAbs in tumors in vivo, we tested NSCLC xenograft models. These models were chosen because nearly 50% of NSCLCs are PD-L1 positive, and PD-L1 IHC is used as a predictive biomarker for NSCLC patients receiving immune checkpoint therapy (Mansfield et al., 2016). NOD scid gamma mice bearing H226 and HCC827 cell-derived xenografts, which exhibit low and moderate PD-L1 expression, respectively (Figure 36A), were treated with a single dose of intravenously administered AtzMab (20 mg / kg, 24 h). 64 PET images acquired 2 hours after Cu]WL12 injection showed significant differences in HCC 827 tumors compared with H226. 64 Cu]WL12. There was a clear reduction in the accumulation of radioactivity in the tumors of AtzMab-treated mice, indicating a reduction in the level of available PD-L1 sites compared to the treated controls (Figures 35A and 35B). The PET imaging results were further confirmed by ex vivo measurements of biodistribution (Figures 35D and 35E, Figures 36B and 36C), which showed a higher [Cu]WL12 accumulation per gram of injected dose in AtzMab-treated mice compared to saline controls. 64 Cu]WL12 showed a significant decrease in percent (%ID / g): 34% in mice with H226 (P<0.0001) and 47% in HCC827 xenografts (P<0.001). PD-L1 expression levels were confirmed by PD-L1 IHC of the xenografts (Figures 35C and 35F). Results showed that [ 64 We demonstrate that [Cu]12 can be used to quantify in vivo targeting of PD-L1 in tumors by AtzMab.

[0123] To evaluate the effect of a single dose of AtzMab on targeting different PD-L1 levels in tumors, PET and biodistribution studies were performed in tumors derived from the CHO-hPDL1 cell line, which has 4-10 times higher PD-L1 expression than NSCLC cells (Figure 36). CHO-hPDL1 / CHO tumor-bearing mice treated with AtzMab (20 mg / kg, 24 hours) showed significantly higher PD-L1 expression in CHO-hPDL1 tumors compared to controls. 64 Biodistribution studies showed a significant decrease in [Cu]WL12 uptake in CHO-hPDL1 tumors compared to AtzMab-treated tumors (Figure 35G). 64 Cu]WL12 binding by 77% (Figure 35H, Figure 36D) (P<0.0001), demonstrating the measurement of tumor PD-L1 targeting by the AtzMab. 64 Cu]WL12 uptake was observed, which was similar to that in hPD-L1 tumors treated with AtzMab. These observations were confirmed by the observation of strong and weak immunoreactivity in hPD-L1 and CHO tumors, respectively (Figure 35I). The results showed that [ 64 Cu]WL12-PET can detect graded levels of PD-L1 expression in tumors, demonstrating that a single 20 mg / kg AtzMab dose can engage a wide range of PD-L1 levels in tumors.

[0124] 3.3.3. Quantification of Dynamic Changes in PD-L1 Expression. PD-L1 is known to be upregulated in response to various cytokines, particularly interferon gamma (IFNγ), which contributes to the dynamic and spatiotemporal heterogeneity in PD-L1 expression (Taube et al., 2015; Taube et al., 2012). We assessed the robustness of [64Cu]WL12 to quantify inducible PD-L1 expression in tumors in vivo and demonstrated that blockade of such upregulated PD-L1 by AtzMab treatment [ 64 We determined whether WL12-PET could monitor the progression of IL-12 expression in the IL-12 cells by [Cu]WL12-PET (Figures 37A, 37B, 37C, 37D, 37E, and 37F).

[0125] To do so, we generated an A549 NSCLC cell line (A549-iPD-L1) with doxycycline-inducible PD-L1 expression. A549 is a Kras G12S lung adenocarcinoma cell line that expresses low PD-L1 at baseline. It was transduced with PD-L1 in the all-in-one lentiviral pINDUCER20 vector (Meerbrey et al., 2011), selected with G418, and PD-L1 induction was confirmed by flow cytometry (Figure 37A), and used for in vitro and in vivo studies. Binding of Cy5-PD-L1-mAb to doxycycline-treated A549-iPDL1 cells was blocked by WL12, demonstrating its specificity (Figure 37B). Furthermore, the cell [ 64 Incubation with [Cu]WL12 showed a 5.5-fold increase in radioactivity uptake in doxycycline-treated versus untreated cells and in PD-L1-low A549 cells (P<0.0001). 64 Cu]WL12 binding was significantly reduced (65%, P>0.0001) in the presence of 60 nM AtzMab, AveMab, and DurMab (Figure 37C). These in vitro studies demonstrated that [Cu]WL12 binding in A549-iPDL1 NSCLC tumors after 72 hours of doxycycline treatment. 64 This was confirmed by in vivo studies showing that the accumulation of [Cu]WL12 in tumors was 65% higher than in A549 control tumors (P>0.0001). 64 Cu]WL12 uptake increase 64 As quantified by [Cu]WL12-PET and biodistribution studies, PD-L1 expression was reduced by >75% in the 20 mg / kg AtzMab-treated group compared to control A549 tumors (Figures 37D and 37E). IHC analysis of tumors showed a strong PD-L1 signal in A549-iPDL1 but not A549 tumors, confirming the imaging and biodistribution results (Figure 37F). Collectively, these results demonstrate the ability to detect dynamic changes in PD-L1 expression levels. 64"Our results demonstrate the potential of [Cu]WL12 and its blockade by AtzMab. PET is therefore expected to play an important role in quantifying dynamic changes in PD-L1 expression within standard clinical workflows, providing a novel method for informing treatment decisions."

[0126] 3.3.4. Quantification of tumor PD-L1 engagement with different antibodies. Radiolabeled anti-PD-L1 antibodies have been developed, and their potential to noninvasively assess PD-L1 expression in human tumor xenografts and syngeneic mouse tumor models has been demonstrated (Chatterjee et al., 2016; Heskamp et al., 2015; Maute et al., 2015; Deng et al., 2016; Hettich et al., 2016; Josefsson et al., 2016). Although such radiolabeled antibody conjugates are currently used clinically to detect PD-L1 (NCT02453984), image other tumor-specific proteins (Gebhart et al., 2016), and determine antibody kinetics, their routine clinical application is limited. To enhance contrast and lesion detection (Pandit-Taskar et al., 2015; Oosting et al., 2016), radiotracers with faster clearance times (hours vs. days) are needed (Wu, 2014). A further limitation is that observations made with radiolabeled antibodies are highly specific to the antibody under investigation and depend on antibody properties such as valency, shape, size, isoelectric point, and dosage, each of which influences its pharmacokinetics. Such unique biophysical characteristics of mAbs also affect plasma half-life, tissue exposure, and ultimately efficacy. New approaches are needed that (i) describe target engagement of PD-L1 antibodies, (ii) take mAb properties into account, and (iii) are applicable to all antibodies.

[0127] To quantify non-invasive PD-L1 engagement in tumors by each of three FDA-approved antibodies, AtzMab, AveMab, and DurMab [ 64We evaluated the capability of [Cu]WL12-PET. MDAMB231 tumor-bearing NSG mice were treated with AtzMab, AveMab, or DurMab, and 24 hours later, they were [ 64 Cu]WL12-PET (Figures 39A, 39B, 39C, and 39D). All treated mice had lower signal in the tumors compared to saline controls, confirming low levels of free PD-L1 from tumor PD-L1 engagement and radiotracer blockade by the mAb. Ex vivo quantification of tumors confirmed these findings, with [Cu]WL12 in tumors of mAb-treated mice at 120 minutes post-injection. 64 The results demonstrated that tumor PD-L1 uptake by [Cu]WL12 was approximately 60% less compared to the saline control (Figure 39E). IHC analysis of the saline control showed moderate to high PD-L1 intensity in the tumor (Figure 39F). The results demonstrate that tumor PD-L1 engagement by PD-L1 therapeutic mAbs is consistent with the [Cu]WL12 uptake by the saline control (Figure 39F). Despite the different biophysical properties, plasma, and tissue kinetics of each antibody, [Cu]WL12 uptake by the saline control (Figure 39E) was approximately 60% less compared to the saline control (Figure 39F). IHC analysis of the saline control showed moderate to high PD-L1 intensity in the tumor (Figure 39F). The results demonstrate that tumor PD-L1 engagement by PD-L1 therapeutic mAbs is consistent with the [Cu]WL12 uptake by the saline control (Figure 39E). 64 We have demonstrated that quantification of WL12-PET is possible.

[0128] 3.3.5. Effect of Dose on PD-L1 Occupancy in Tumors. Antibody dynamics in tumors are governed by both intrinsic and extrinsic tumor parameters (Agoram, 2009). Recently, it was discovered that factors other than PD-L1 expression itself can reduce the accumulation of the PD-L1-targeted therapeutic AtzMab and its mouse chimera (PRO304397) in NSCLC, TNBC, and colon tumors (Chatterjee et al., 2016). Furthermore, at doses below 1 mg / kg, systemically injected radiolabeled anti-PD-L1 antibody PRO304397 primarily associated with the tumor vasculature and showed minimal diffusion into the tumor parenchyma in a PD-L1-expressing syngeneic mouse tumor model (Deng et al., 2016). These findings may be due to factors such as increased interstitial pressure within the tumor (Baxter et al., 1989; Baxter et al., 1990), which prevents mAb accumulation in tumors, contributing to resistance (Goel et al., 2011). Such effects may also hinder the access of large PD-L1-directed agents to targeted tumor cells and immune infiltrates.Measurements of PD-L1 and PD-1 therapeutic occupancy have not been reported in tumors and are limited to evaluations using PBMCs.

[0129] To evaluate the effect of dose on tumor PD-L1 occupancy in tumors, MDAMB231 tumor-bearing mice were injected with increasing doses of AtzMab, ranging from 0.009 to 24 mg / kg body weight. After 24 hours, [ 64 Imaging and biodistribution studies were performed 2 hours after injection of [Cu]WL12. PET images of mice receiving 0.06 mg / kg showed significant differences compared to untreated controls. 64 There was no difference in [Cu]WL12 uptake, indicating low PD-L1 occupancy by AtzMab in the tumor (Figure 41A). The 0.6 and 3.2 mg / kg doses showed a proportional decrease in signal intensity in the tumor, with the 3.2 mg / kg dose demonstrating nearly 100% target engagement by the antibody in the tumor.

[0130] The radioactivity accumulated in the tumor (%ID / g) was then used to calculate the inhibitory sigmoid E maxThe model was fitted. The %ID / g data are based on the dose of AtzMab used in our experiments and the peptide radiotracer [ 64 The relationship between the reduction of free PD-L1 ligand in tumors detected using [Cu]WL12 and 50% of maximal PD-L1 engagement in tumors (ID ) was well-fitted and explained (Figures 41B and 41C). 50 ) or maximum fractional reduction of free PD-L1 ligand from baseline (I max The dose of AtzMab responsible for the inflammatory bowel disease was estimated to be 0.43 mg / kg (Table 2). max ID involved in 90% and 96% of 90 and ID 96 These dose levels corresponded to 0.87 mg / kg and 1.19 mg / kg, respectively. These dose levels are comparable to the 1 mg / kg dose reported by Denget et al. for PRO304397 (Deng et al., 2016). Similar mean V ss Assuming that the dose is 50 mL / kg, the ID 50 , ID 90 and ED 96 The expected mean plasma concentrations resulting from these were tentatively estimated to be 59 nM (8.6 mcg / mL), 120 nM (17.4 mcg / mL), and 164 nM (23.8 mcg / mL), respectively. These results demonstrate the feasibility of using measurements made in the tumor for dose selection and optimization.

[0131] The interaction of antibodies with their targets differs from that of small molecules in that antibody binding can affect the natural dynamics of PD-L1, such as PD-L1 stabilization or internalization and the development of anti-therapeutic antibodies, which can have significant impacts on the tumor and serum kinetics of antibodies (Tabrizi et al., 2006). Early pharmacokinetic studies of AtzMab reported nonlinear PK at doses of 0.6-1 mg / kg and linear PK at doses above 1 mg / kg, with a trend toward decreased serum antibody concentrations in patients who developed ATA (Stroh et al., 2017). However, the impact of such tumor-intrinsic and -extrinsic parameters on the PK and occupancy of PD-L1 antibodies in tumors is unknown.

[0132] [Table 2]

[0133] Detecting temporal changes in antibody dynamics in tumors 64 To investigate the capabilities of [Cu]WL12-PET, NSG mice bearing MDAMB231 tumors were injected with AtzMab at doses of 0.6 and 10 or 20 mg / kg, which produce nonlinear and linear kinetics, respectively, and PET imaging and biodistribution studies were performed at 24 and 120 hours. At 24 hours, the tumor uptake values ​​were also reflected in [Cu]WL12-PET in all three dose groups compared to untreated controls. 64 At 120 hours, there was a significant decrease in [Cu]WL12 uptake in the 0.6 mg / kg dose group compared to 24 hours (Figures 41D and 41E). 64 In contrast, in the 10 or 20 mg / kg treatment groups, [Cu]WL12 uptake was significantly increased. 64 There was no significant time difference in [Cu]WL12 uptake. 64 Cu]WL12 uptake was similar in the 0.06 mg / kg treated and saline control groups, suggesting drug clearance from the tumor and reflecting nonlinear PK of AtzMab at low doses. Results show that in a mouse model, both dose- and time-dependent changes in PD-L1 engagement were observed. 64Cu]WL12-PET.

[0134] 3.3.6. Discussion Immune checkpoint therapeutics are being tested in hundreds of clinical trials, approximately 25% of which target PD-L1. Because only 30% of patients receiving PD-L1 therapy respond to treatment, the molecular and cellular basis of response and resistance to these therapies are being investigated using transcriptional, genetic, and epigenetic studies. The relationship of dose to drug accumulation and target saturation in relation to efficacy in tumors is unknown. In addition, the large size of antibody therapeutics limits tumor penetration, posing unique challenges for pharmacodynamic evaluation at the site of action. Validated methods that consider both tumor-intrinsic and tumor-extrinsic parameters, provide real-time PD-L1 saturation / occupancy data in tumors, and can be widely applied have been lacking. This lack of knowledge hinders dose selection, dose optimization, treatment development, and treatment optimization to reduce toxicity. Our current study demonstrated that radiolabeled PD-L1-binding peptides can noninvasively detect variable and dynamic PD-L1 expression levels and can be used to measure tumor occupancy while taking into account tumor-intrinsic parameters (PD-L1 expression, recycling, interstitial pressure) and extrinsic parameters (antibody isotype, kinetics, ATA, catabolism), thus providing a universal means for monitoring the therapeutic activity of PD-L1:PD-1 interaction-blocking PD-L1 antibodies in tumors.

[0135] Although IHC-based clinical trials have been previously developed to assess PD-L1 expression in tumors (Herbst et al., 2014; Roach et al., 2016; Meng et al., 2015), PD-L1 IHC only accounts for a small fraction (0.1%) of single lesions. Such approaches have significant limitations because PD-L1 expression in the tumor microenvironment is spatially and temporally heterogeneous, and immunotherapy responses are inherently delayed, complex, and abscopal. Furthermore, tissue samples obtained by biopsy for testing are typically very limited and may be required for molecular profiling to identify targetable oncogenic mutations in other pathways (e.g., BRCA1, BRCA2, PARP) that confer sensitivity or resistance to existing therapies (Nolan et al., 2017). Such precious samples often make it impractical to perform multiple PD-L1 assessments for reliable delineation of PD-L1 expression (Gibney et al., 2016). These issues are exacerbated in patients with metastatic disease, a population in which immune checkpoint therapies have been extensively studied. Such factors contribute to our limited success in advancing immunotherapy. The dynamic nature of both PD-L1 expression and the broader tumor immune microenvironment necessitates the development of PET radiotracers that enable rapid assessment of the TME. 64 The disclosed studies using [Cu]WL12 demonstrate that variable and dynamic changes in PD-L1 expression can be quantified within a standard clinical workflow, with important clinical implications for patient selection and monitoring therapy.

[0136] PD-L1 therapeutic antibodies have become important agents in cancer immunotherapy. For small molecules, in vitro binding affinity measurements and occupancy studies are routinely used for dose selection and pharmacological response prediction in CNS diseases (Lee et al., 2006). However, large molecules such as antibodies pose unique challenges in predicting in vivo receptor occupancy based on in vitro binding affinity (Agoram, 2009). Antibody concentrations in tumors are influenced by several tumor-intrinsic parameters, such as antigen density and turnover, tumor burden, and tumor perfusion, which limits mAb penetration into tumors. mAb tumor and plasma concentrations are further influenced by tumor-extrinsic factors, such as affinity, dose, patient heterogeneity, cachexia, and the development of anti-therapeutic antibodies (Sheng et al., 2017). Existing PK / PD prediction models rely on in vitro and PBMC-based measurements to predict optimal doses (Deng et al., 2016). However, the presently disclosed subject matter demonstrates that PET can be used to measure PD-L1 occupancy by therapeutic antibodies noninvasively in tumors in real time.

[0137] Radiolabeled antibodies such as atezolizumab, supported by peripheral pharmacodynamic assessments and PK / PD modeling, are routinely used to predict the mAb dose levels required to achieve desired PD-L1 occupancy in tumors (Deng et al., 2016). Because antibody plasma and tumor concentrations are influenced by the antibody isotype and biophysical properties such as charge and valency, occupancy predictions derived from these measurements and mathematical modeling are often specific to a given antibody; therefore, generalizability of such observations to other PD-L1 mAbs is limited (Kamath, 2016). There is a need for tools that can be used to assess antibody kinetics and tumor target engagement for the ever-expanding number of therapeutic PD-L1 mAbs. The subject matter of this disclosure addresses this need. In silico modeling studies combined with in vitro and in vivo data using WL12-PET have demonstrated the feasibility of quantifying PD-L1 saturation / occupancy in tumors, a concept applicable to all therapeutic PD-L1 mAbs in clinical trials.

[0138] Taken together, the data disclosed herein demonstrate that the dynamic changes in tumor PD-L1 expression and PD-L1 saturation / occupancy by therapeutic antibodies can be quantified noninvasively by considering two features: independent of antibody characteristics and tumor intrinsic and extrinsic parameters. The results disclosed herein relating dose to tumor PD-L1 occupancy for three different therapeutic antibodies, AtzMab, AveMab, and DurMab, are expected to be relevant to treatment response and dosing efficacy.

[0139] 3.3.7. Overview. The presently disclosed subject matter demonstrates that radiolabeled PD-L1-binding peptides can noninvasively detect variable and dynamic PD-L1 expression levels and can be used to measure tumor occupancy while taking into account tumor-intrinsic parameters (PD-L1 expression, recycling, interstitial pressure) and extrinsic parameters (antibody isotype, kinetics, ATA, catabolism), thus providing a universal means to monitor the therapeutic activity of PD-L1:PD-1 interaction-blocking PD-L1 antibodies in tumors.

[0140] [ 64 Studies with Cu]WL12 demonstrate that variable and dynamic changes in PD-L1 expression can be quantified within a standard clinical workflow, with important clinical implications for patient selection and monitoring therapy.

[0141] Existing PK / PD prediction models for antibodies rely on in vitro and PBMC-based measurements to predict optimal doses (Deng et al., 2016). However, the presently disclosed subject matter demonstrates that PET can be used to measure PD-L1 occupancy by therapeutic antibodies noninvasively in tumors in real time.

[0142] There is a need for tools that can be used to assess the antibody kinetics and tumor target engagement potential of the ever-expanding array of PD-L1 therapeutic mAbs. The subject matter of the present disclosure addresses this need. In silico modeling studies combined with in vitro and in vivo data using WL12-PET demonstrate that PD-L1 saturation / occupancy in tumors can be quantified, a concept that can be applied to all PD-L1 therapeutic mAbs in clinical trials.

[0143] (References) All publications, patent applications, patents, and other references mentioned herein are indicative of the level of skill of those skilled in the art to which the subject matter of this disclosure pertains. All publications, patent applications, patents, and other references (e.g., websites, databases, etc.) mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although references to numerous patent applications, patents, and other references are made herein, it is understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art. 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Sequence Listing: SEQ ID NO: 1 WL12 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Leu-Hyp-Trp-Ser-Trp(methyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2) SEQ ID NO: 2 DK-A-221 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2

Claims

1. A compound selected from the group consisting of compounds of formula (I) and formula (II): 【Chemistry 1】 DK-A-221-(L) ｎ -Rpt (II)、 wherein DK-A-221 is cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH 2 ; (In the formula: n is an integer selected from the group consisting of 0 and 1; L is the group of the compound of formula (I) １３ ornithine (Orn) primary amine group, or a compound of formula (II) １３ a linker attached to a lysine (Lys) primary amine group; Rpt is the reporting site; wherein when n is 1, the reporting moiety is attached to the linker, and when n is 0, the reporting moiety is attached to the linker of the compound of formula (I). １３ ornithine (Orn) primary amine group, or the compound of formula (II) １３ linked to a lysine (Lys) primary amine group; and wherein the reporting moiety is selected from the group consisting of a radiometal-containing chelator, a fluorescent dye, a photoacoustic reporting molecule, and a Raman-active reporting molecule.

2. 2. The compound of claim 1, wherein the reporting moiety is a chelator comprising a radioactive metal, and the chelator is selected from the group consisting of DOTAGA (1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTASA (1,4,7,10-tetraazacyclododecane-1-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A (10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2-(bis-carboxymethylamino)-ethyl]- 4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), TCMC (2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)-cyclododecane), oxo-DO3A (1-oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NH ２ -Bn-oxo-DO3A (1-oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((1,8-N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane)), MM-TE2A, DM-TE2A, CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11-tetraaza cyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid)), CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODA (1,4,7-triazacyclononane-1,4-diacetate); NODAGA (1,4,7-triazacyclononane-1,4-diacetate); N,N',N'',1-glutaric acid-4,7-acetic acid), (NOTAGA) 1,4,7-triazonane-1,4-diyl)diacetic acid DFO (desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-™ (N,N',N'',tris(2-mercaptoethyl)-1,4,7-triazacyclononane), Diamsal (1,8-diamino-3,6,10,13,16,19-hexaazanoyl)acetate Zabicyclo(6,6,6)eicosane, 3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), Sarar (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19-hexazabicyclo[6.6.6]icosan-1-ylamino)methyl)benzoic acid), and BaBaSar.

3. 10. The compound of claim 1, wherein the reporting moiety is a chelator containing a radiometal, and the chelator is selected from the group consisting of: 【Chemistry 2】

4. 2. The compound of claim 1, wherein the reporting moiety is: (a) ９４ｍ Tc, ９９ｍ Tc, １１１ In, ６７ Ga, ６８ Ga, ８６ Y. ９０ Y. １７７ Lu, １８６ Re, １８８ Re, ６０ Cu, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ５５ Co, ５７ Co, ４７ Sc, ２２５ Ac, ２１３ Bi, ２１２ Bi, ２１２ Pb, １５３ Sm, １６６ Ho, １５２ Gd, ８２ Rb, ８９ Zr, １６６ Dy and ２１１ a chelating agent comprising a radioactive metal selected from the group consisting of At; (b) a fluorescent dye, wherein the fluorescent dye is selected from the group consisting of carbocyanine, indocarbocyanine, oxacarbocyanine, thicarbocyanine, merocyanine, polymethine, coumarin, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY) dyes, Cy5, Cy5.5, Cy7, and VivoTag-680. , VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFlu or750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IR Dye 800, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS; (c) a photoacoustic reporting molecule, wherein the photoacoustic reporting molecule is selected from the group consisting of a dye or a nanoparticle; wherein the dye comprises a fluorescent dye selected from the group consisting of indocyanine green (ICG), Alexa Fluor 750, Evans Blue, BHQ 3, QXL 680, IRDye 880CW, MMPSense 680, methylene blue, PPCy-C8, and Cypate-C18; and wherein the nanoparticle is selected from the group consisting of a plasmonic nanoparticle, a quantum dot, a nanodiamond, a polypyrrole nanoparticle, a copper sulfide nanoparticle, a graphene nanosheet, an iron oxide-gold core-shell nanoparticle, a Gd ２ O ３ nanoparticles, single-walled carbon nanotubes, dye-loaded perfluorocarbon nanoparticles, and superparamagnetic iron oxide nanoparticles; and (d) a Raman-active reporting molecule, wherein the Raman-active reporting molecule is selected from the group consisting of a single-walled carbon nanotube (SWNT) and a surface-enhanced Raman scattering (SERS) agent; wherein the SERS agent comprises a metal nanoparticle labeled with a Raman-active reporter molecule; and wherein the Raman-active reporter molecule comprises a fluorescent dye; and wherein the fluorescent dye is selected from the group consisting of Cy3, Cy5, rhodamine, and chalcogenopyrylium dyes.

5. 2. The compound of claim 1, wherein the linker and reporting moiety combination (Rpt-L) is selected from the group consisting of: (a) 【Transformation 3】 (In the formula: Rpt is the reporting site; W １ is C １ -C ６ Alkylene, C ３ -C ６ selected from the group consisting of cycloalkylene, and arylene; W ２ is -NR １ -(C=O)-, -NR １ -(C=S)-, -(C=O)-NR １ -, -(C=S)-NR １ -, and -S-, wherein each R １ are independently H or C １ -C ４ is alkyl; Each R ２ are independently H or -COOR ３ where each R ３ are independently H, C １ -C ６ Alkyl, C ２ -C １２ Aryl or C ４ -C １６ alkylaryl; b is an integer selected from the group consisting of 0, 1, 2, and 3; d is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, and 8; and where the wavy line indicates the point of attachment between the linker and the peptide; (b) Rpt-X-Y-Z-W ３ - (In the formula: Rpt is the reporting site; X and Z are each independently C １ -C ８ Alkyl, C ２ -C ８ Alkenyl, C ２ -C ８ Alkynyl, C １ -C ８ Heteroalkyl, C ２ -C ８ Heteroalkenyl, C ２ -C ８ Heteroalkynyl, C １ -C ８ alkoxy, or a bond, each of which is 0 to 5 R Ａ may be substituted with; Y and W ３ are each independently -O-, -S(O) ｐ -, -NH-, -NR Ｂ -, -CH=CH-, -CR Ｂ =CH-, -CH=CR Ｂ -, -NH-CO-, -NH-CO ２ -, -NR Ｂ -CO-, -NR Ｂ -CO ２ -, -CO-NH-, -CO ２ -NH-, -CO-NR Ｂ -, -CO ２ -NR Ｂ - or bond; p is 0, 1, or 2; R Ａ represents, at each occurrence, halogen, hydroxy, amino, cyano, nitro, CO ２ H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted mono- or di-alkylamino, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted mono- or di-alkylcarboxamido, optionally substituted aryl, or optionally substituted heteroaryl; R Ｂ is, at each occurrence, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted mono- or di-alkylamino, optionally substituted alkylthio, optionally substituted aryl, or optionally substituted heteroaryl; or (c) Rpt-(amino acid).

6. 2. The compound of claim 1, wherein the reporting moiety is ６４ a DOTAGA chelator containing a Cu radiometal; and a compound of formula (I): 【Chemistry 4】

7. 10. A composition for an imaging method comprising the compound of claim 1, wherein the imaging method is an imaging method for detecting programmed death-ligand 1 (PD-L1), comprising the steps of: (a) providing to a subject an effective amount of a compound of claim 1; (b) contacting one or more cells or tissues with said compound; (c) Creating an image and detecting PD-L1.

8. 8. The composition for the imaging method of claim 7, wherein contacting the one or more cells or tissues with the compound is performed in vitro, in vivo, or ex vivo.

9. 9. The composition for the imaging method of claim 8, wherein the contacting of the one or more cells or tissues with the compound is performed in a subject.

10. 10. The composition for the imaging method of claim 9, wherein the subject is a human, rat, mouse, cat, dog, horse, sheep, cow, monkey, bird, or amphibian.

11. 8. The composition for the imaging method of claim 7, wherein the detection of PD-L1 occurs within 60 to 120 minutes after administration of the compound to the subject.

12. 8. The composition for an imaging method according to claim 7, wherein the imaging method is used to detect: (a) cancer, wherein the cancer is selected from the group consisting of blastoma, carcinoma, glioma, leukemia, lymphoma, melanoma, myeloma, sarcoma, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colorectal cancer, esophageal cancer, gastric cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary tract cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, renal cancer, bladder cancer, brain tumor, adenoma, and metastatic cancer; (b) a solid tumor, wherein the solid tumor is located in an organ selected from the group consisting of the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovary, cervix, stomach, rectum, bladder, uterus, testicle, and pancreas; (c) infectious diseases, including microbial infections, wherein the microbial infection is selected from the group consisting of infections caused by one or more microorganisms selected from the group consisting of Mycobacterium tuberculosis, Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus spp., including Staphylococcus aureus and coagulase-negative staphylococci, Enterococcus spp., Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides spp., Acinetobacter spp., Helicobacter spp., Candida spp., methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecium (VRE); (d) inflammation, wherein the inflammation is associated with a disorder selected from the group consisting of asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis media, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, lupus, systemic lupus erythematosus, and vasculitis, or wherein the inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus; (e) one or more immune cells in the tumor; (f) systemic distribution of immune cells within a tumor or subject; (g) immune cell responses to infection; (h) immune cell responses in tumors or normal tissues to inflammatory diseases; (i) PD-L1 expression level; or (j) Occupancy or target engagement of PD-L1 by antibodies, peptides, or small molecular weight agents at the tumor site or in normal tissues.

13. A kit for detecting programmed death-ligand 1 (PD-L1), comprising the compound of claim 1.

14. 2. The compound of claim 1, wherein the compound is a compound of formula (II), n is 0, and the reporting moiety is Al. １８ and a NODAGA chelator containing F, and said compound has the following structure: 【Transformation 5】

15. 2. The compound of claim 1, wherein the compound is a compound of formula (II), n is 0, and the reporting moiety is Al. １８ and a DOTA chelator containing F, and said compound has the following structure: 【Transformation 6】

16. 2. The compound of claim 1, wherein the compound is a compound of formula (II), n is 0, and the reporting moiety is Al. １８ and a DOTAGA chelator containing F, and said compound has the following structure: 【Transformation 7】

17. 10. The compound of claim 1, wherein the compound has the following structure: 【Transformation 8】 where R is a reporting moiety that includes a NODA or NOTA chelator containing a radiometal.

18. 2. The compound of claim 1, wherein the compound is selected from the group consisting of compounds of formula (I) and formula (II): 【Chemistry 9】 DK-A-221-(L) ｎ -Rpt (II)、 wherein DK-A-221 is cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH 2 ; (In the formula: n is an integer selected from the group consisting of 0 and 1; L is the group of the compound of formula (I) １３ ornithine (Orn) primary amine group, or a compound of formula (II) １３ a linker attached to a lysine (Lys) primary amine group; and Rpt is a reporting moiety comprising a chelator containing a radiometal, wherein the chelator is selected from the group consisting of DOTAGA (1,4,7,10-tetraazacyclododecane, 1-(glutaric acid)-4,7,10-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTASA (1,4,7,10-tetraazacyclododecane-1-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A (10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA (7-[2-(bis-carboxymethylamino)-ethyl]-4,10 -bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid), 3p-C-DEPA (2-[(carboxymethyl)[5-(4-nitrophenyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), TCMC (2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)-cyclododecane), oxo-DO3A (1-oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid), p-NH ２ -Bn-oxo-DO3A (1-oxa-4,7,10-tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A ((1,8-N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane)), MM-TE2A, DM-TE2A, CB-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11-tetraazabicyclo[6.6.2]hexadecane), Chlotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid), CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODA (1,4,7-triazacyclononane-1,4-diacetate); NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid), (NOTAGA) 1,4,7-triazonane-1,4-diyl)diacetic acid DFO (desferoxamine), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}-acetic acid), TACN-™ (N,N',N'',tris(2-mercaptoethyl)-1,4,7-triazacyclononane), Diamsal (1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[4.2.1.2 ... chloro(6,6,6)eicosane, 3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), Sarar (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine), AmBaSar (4-((8-amino-3,6,10,13,16,19-hexazabicyclo[6.6.6]icosan-1-ylamino)methyl)benzoic acid), and BaBaSar; and wherein the reporting moiety is attached to a primary amine group of an amino acid of a peptide comprising a compound of formula (I) or formula (II).

19. 20. The compound of claim 18, wherein said compound comprises a compound of formula (II) and said reporting moiety is NODA, wherein said NODA comprises a radiometal.

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