Tumor and immune cell imaging based on PD-l1 expression

Abstract

To provide a contrast agent for detecting Programmed Death Ligand 1 (PD-L1), which is expressed on the surface of cells in a subject and the like, with high sensitivity and which is cleared within a short period of time, as well as an imaging method for detecting PD-L1.SOLUTION: An imaging agent has a peptide residue that specifically binds to PD-L1, preferably represented by the following formula (I) [n is 0 or 1; L is a linker; Rpt is a reporting moiety represented by DOTAGA (1,4,7,10-tetraazacyclododecane) coordinated with a 64Cu radioactive metal].SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is incorporated by reference in its entirety. No. 62 / 438,575, filed on June 14, 2017. The benefit of filed U.S. Provisional Patent Application No. 62 / 519,534 is claimed. Federally funded research and development

[0002] This invention was made in part through funding from the National Institutes of Health (NIH) awarded NIH R01CA1663 This invention was made with government support under the Patent Application No. 10 / 1999 / 010010. The government has certain rights in this invention. [Background technology]

[0003] Molecular imaging reports on the status of the tumor immune microenvironment and predicts the efficacy of immunomodulatory therapies. This can guide immunotherapy strategies to enhance immune responses. There are few contrast agents that can be used.

[0004] Immunotherapy, which suppresses one's own immune system to kill cancer cells, is a key treatment for various cancers. (Topalian et al., 2016). Significantly improved treatment outcomes Despite these results, many cancers do not respond to immunomodulatory therapy. Existing companion diagnostics that act via immunotherapy provide only a snapshot of the dynamic tumor-immune environment. provide a predictive model for treatment response, and often do not accurately predict treatment response (Mansfield and Do Non-invasive imaging techniques offer quantitative, real-time insight into tumor biology. It can provide assessment and guide drug development (Willmann et al., 2008).

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

[0006] Programmed death-ligand 1 (PD-L1) is an immune-mediated cytokine that is overexpressed in several cancers. PD-L1 is a checkpoint protein that contributes to tumor immunosuppression. , demonstrating tumor response to PD-1 and PD-L1 targeted therapy. Non-invasive PD in human tumor xenografts and syngeneic tumor models using the L1 antibody -L1 expression can be assessed ( 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 Conjugates are increasingly being used for imaging tumor-specific proteins, but Long clearance times of up to several days for improved trust and lesion detection required (Pandit-Taskar et al., 2015; Oosting et al., 2016 ). Summary of the Invention

[0007] In some aspects, the presently disclosed subject matter provides a method for the treatment of cancers caused by the activation of PD-L1. a conjugate of a peptide having binding specificity to a reporting moiety, and optionally Imaging agents comprising a linker, the linker, if present, separating the peptide from the reporter If no linker is present, the reporting moiety will be attached to the peptide. The present invention provides an imaging agent that is directly attached to a peptide via the primary amine of an amino acid. In such cases, the reporting moiety is incorporated directly into the peptide, e.g., The binding moiety is a radiolabel of the peptide, such as radiolabeled iodotyrosine or fluorotyrosine. Contains amino acids.

[0008] In certain embodiments, the peptide that has binding specificity for PD-L1 is an amino acid sequence of PD-L1. It interacts with amino acids Y56, E58, A113, M115, and Y123.

[0009] In certain embodiments, the peptide is WL12 and the imaging agent is a compound represented by Formula (I), Formula (II), and and a compound selected from the group consisting of 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 site; and The anchor, when present, is a pen containing an imaging agent of formula (I), formula (II), or formula (III). (attached to the primary amine group of an amino acid in the peptide).

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

[0011] In another aspect, the presently disclosed subject matter provides a method for the treatment of a cell death by a cell transplant using a programmed death-ligand 1 (PD-1), comprising the steps of: (a) Imaging methods for detecting programmed death ligand 1 (L1): Conjugation of a peptide with binding specificity for PD-L1 with a reporting moiety and optionally a linker, wherein the linker, if present, The linker connects the peptide to the reporting moiety, and in the absence of a linker, the reporting moiety Imaging agents in which the binding moiety is directly attached to the peptide via the primary amine of an amino acid of the peptide. (b) contacting one or more cells or tissues with an imaging agent; (c) imaging to detect PD-L1. is a compound of formula (I), or Y56, E58, A113, M11 of PD-L1 5 and Y123.

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

[0013] In yet a further aspect, the presently disclosed subject matter provides a method for treating a cancer cell line comprising administering to a patient a therapeutically effective amount of ... The present invention provides a kit for detecting a PD-L1-specific antibody against a PD-L1-specific antibody. a conjugate of a peptide having binding specificity to a reporting moiety, and optionally and a linker, if present, between the peptide and the reporter. If no linker is present, the reporting moiety is linked to the peptide. The present invention includes an imaging agent directly attached to a peptide via the primary amine of an amino acid of the formula:

[0014] Certain aspects of the presently disclosed subject matter have been described above and are generally referred to by the presently disclosed subject matter. The accompanying examples are provided in part or in part as best explained herein below. Other aspects will become apparent as the description proceeds and in conjunction with the examples and figures. [Brief explanation of the drawings]

[0015] Having thus described the subject matter of the present disclosure in general terms, reference will now be made to the accompanying drawings, in which: , 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 Figures 34B and 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, including any drawings, are available upon request and the necessary fee. will be provided by the Office on payment of DETAILED DESCRIPTION OF THE INVENTION

[0017] The subject matter of the present disclosure is described more fully below with reference to the accompanying drawings, in which: Some, but not all, embodiments of the illustrated subject matter are shown. The subject matter of this disclosure may be embodied in many different forms, and the terms "subject matter" and "subject matter" refer to the same element. The present invention should not be construed as being limited to the embodiments set forth in the specification; rather, these embodiments is provided so that this disclosure will satisfy applicable legal requirements. Many modifications and other embodiments of the presently disclosed subject matter will become apparent to those skilled in the art to which the presently disclosed subject matter pertains. This invention will come to mind to one skilled in the art having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, the subject matter of this disclosure should not be limited to the particular embodiments disclosed. However, modifications and other embodiments are intended to be within the scope of the appended claims. I want you to understand that.

[0018] I. Compositions Comprising Contrast Agents In some embodiments, the presently disclosed subject matter is directed to immune checkpoint transcription factors such as PD-L1. Highly specific peptide-based positron emission tomography (PET) for protein detection These imaging agents can be used to specifically and quantitatively assess tumor PD-L1 expression. and can be detected immediately after administration to a subject.

[0019] Thus, in some embodiments, the presently disclosed subject matter provides a method for treating apoptosis with programmed death ligand 1 (P Conjugation of a peptide with binding specificity for the nucleotide sequence D-L1 with a reporting moiety. and optionally a linker, wherein the linker, if present, is a peptide. The linker connects the reporting moiety to the nucleotide sequence, and in the absence of a linker, the reporting moiety is Contrast agents whose moieties are directly attached to peptides via the primary amines of the peptide amino acids In other embodiments, the reporting moiety is incorporated directly into the peptide, e.g. , where the reporting moiety is a radiolabeled iodotyrosine or fluorotyrosine, etc. The peptide contains radiolabeled amino acids.

[0020] In some embodiments, the binding specificity for programmed death-ligand 1 (PD-L1) The peptide having the formula: In this form, the peptide binds to amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, a peptide having binding specificity for PD-L1 can interact with PD-L1. The peptides may interact with five specific amino acids of PD-L1. The peptides target amino acids Y56, E58, A113, M115, and Y123 of PD-L1. In some embodiments, the PD-L1-interacting peptide may be a peptide Peptide WL12 is cyclo-(-Ac-Tyr-NMeAla-As n-Pro-His-Leu-Hyp-Trp-Ser-Trp(methyl)-NMeNle-NM It may have the amino acid sequence of (Nle-Lys-Cys-)-Gly-NH2) (SEQ ID NO: 1). In some embodiments, WL12 can interact with four amino acids of PD-L1. In certain embodiments, WL12 is a PD-L1 polypeptide that mutates amino acids Y56, E58, D61, and In some embodiments, WL12 interacts with PD-L1 5 and A113. In a specific embodiment, WL12 interacts with one amino acid of PD-L1. It can interact with acids Y56, E58, A113, M115 and Y123. In this study, the peptide that interacts with PD-L1 is DK-A-221. 21 is cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-T rp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys- )-Gly-NH2) (SEQ ID NO: 2). In certain embodiments, DK-A-221 may interact with four amino acids of PD-L1. DK-A-221 binds to amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, DK-A-221 interacts with the five amino acids of PD-L1. In a specific embodiment, DK-A-221 interacts with amino acid Y5 of PD-L1. 6, E58, A113, M115, and Y123. The PD-L1 interacting peptide is DK-A-222. DK-A-222 can interact with four amino acids of PD-L1. In certain embodiments, DK-A-222 interacts with amino acids Y56, E58, D61, and A113 of PD-L1. In some embodiments, DK-A-222 binds to the five amino acids of PD-L1. In certain embodiments, DK-A-222 can interact with amino acid Y of PD-L1. 56, E58, A113, M115 and Y123.

[0021] In some embodiments, the peptide with binding specificity for PD-L1 has SEQ ID NO: It may have at least 80% sequence identity to No. 1. Binding specificity to PD-L1 A peptide having the sequence P may have at least 80% sequence identity to SEQ ID NO:2. Peptides having binding specificity for D-L1 have a binding specificity of at least 85% to SEQ ID NO: 1 Peptides with binding specificity for PD-L1 may have a sequence identity of SEQ ID NO: 2. The antibody may have binding specificity for PD-L1. The peptide may have at least 90% sequence identity to SEQ ID NO: 1. A peptide having binding specificity for -L1 has at least 90% binding specificity to SEQ ID NO:2. The peptides with binding specificity for PD-L1 may have sequence identity with the peptides set forth in SEQ ID NO: 1. It may have at least 95% sequence identity to PD-L1. The peptide may have at least 95% sequence identity to SEQ ID NO:2. Peptides with binding specificity for L1 have 100% sequence identity to SEQ ID NO:1. The peptide with binding specificity for PD-L1 may have the sequence: 00% sequence identity.

[0022] The term "percent identity" as known in the art refers to the degree of identity between two or more polypeptides. The relationship between sequences of a given nucleotide or two or more polynucleotide sequences. In the art, "identity" is sometimes determined by a polypeptide or polynucleotide, as determined by a match between strings of identical sequences "Identity" and "similarity" also refer to the degree of sequence relatedness between nucleotide sequences. These values can be readily calculated by known methods, including but not limited to those listed in Computational Molecular Biology (Lesk , AM, ed.) Oxford University Press, N ew York (1988); Biocomputing: Informatics s and Genome Projects (Smith, DW, ed. ) Academic Press, New York (1993); ter Analysis of Sequence Data, Part I (G Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); ce Analysis in Molecular Biology (von He inje, G., ed.) Academic Press (1987); an d Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). The preferred method for determining identity is It is designed to give the best match between sequences. To determine identity and similarity The method is codified in a publicly available computer program. Analysis and percent identity calculations are performed using Lasergene Bioinformatics Computing Suite Megalign program (DNASTAR, Maddie Multiple sequence alignment can be performed using the NIH (Irvine, WI) The Clustal alignment method (Higgins and Sharp (198 9) CABIOS. 5:151-153) to perform pairwise alignment. This can be done using default parameters, including the default parameters.

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

[0024] The terms "naturally occurring amino acid" and "naturally encoded amino acid" are interchangeable. amino acids that are potentially used and encoded by the genetic code, as well as those that are modified after synthesis Amino acids encoded by the genetic code, e.g., hydroxyproline, γ-carbohydrate, xyglutamate, and O-phosphoserine.

[0025] "Amino acid analogs" are compounds that have the same basic chemical structure as naturally occurring amino acids. that is, it has an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. compounds such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs may be prepared by modifying R groups, e.g., norleucine) or modified peptide backbones, but naturally occurring They will retain the same basic chemical structure as amino acids.

[0026] The terms "non-naturally occurring amino acid" and "non-naturally encoded amino acid" are interchangeable. are used interchangeably and have the same basic chemical structure as naturally occurring amino acids, but are present in "Naturally occurring amino acids" refers to compounds that are not incorporated into growing polypeptide chains by "Amino acids" are naturally encoded amino acids (including the 20 standard amino acids, but amino acids resulting from modifications (e.g., post-translational modifications) of the amino acid sequence (including, but not limited to, Amino acids that are not naturally incorporated into growing polypeptide chains by the translation complex These include, but are not limited to, amino acids that can be inserted into a polypeptide sequence. is a list of non-naturally occurring amino acids that can substitute for wild-type residues in a polypeptide sequence. A non-limiting list of examples includes β-amino acids, homoamino acids, cyclic amino acids and derivatized amino acids. These include amino acids with side chains. Examples include (in L or D form; Abbreviated here): citrulline (Cit), homocitrulline (hCit), Nα-methyl N-methyl-citrulline (NMcCit), N-methyl-homocitrulline (N-MeHoCit), or ornithine (Orn), Nα-methylornithine (Nα-MeOrn or NMeO rn), sarcosine (Sar), homolysine (hLys or hK), homoarginine ( hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR) , Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMe HoK). Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), octahydroin Dole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2 -naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Ti c), 2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe ), para-aminophenylalanine (4AmP or 4-amino-Phe), 4-guanidino Phenylalanine (Guf), glycyrrhizin ("K(Nε-glycyl)" or "K( glycyl) or abbreviated as "K(gly)"), nitrophenylalanine (nitr ophe), aminophenylalanine (aminophe or amino-Phe), benzo diphenylalanine (benzylphe), gamma-carboxyglutamic acid (γ-carboxyglutamic acid boxyglu), hydroxyproline (hydroxypro), p-carboxyl phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methylvaline (NM eVal), Nα-methylleucine (NMeLeu), Nα-methylnorleucine (NMe Nle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), Cetylarginine (acetylarg), α,β-diaminopropionic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexyl Alanine (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, piperidine acid, aminocapric acid, aminoheptanoic acid, Minopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethyl ascorbic acid Paragine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo- Isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxybenzoate Hydroxyproline (Hyp). γ-Carboxyglutamate, ε-N,N,N-trimethylsilyl Lysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine lysine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-amino- O-phthalic acid (4APA), N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl Cylaminyl-L-threonine, O-phosphotyrosine and other similar amino acids, and Derivatized forms of any of those specifically listed.

[0027] A "peptide" or "protein" is a series of small molecules joined together by peptide bonds. Contains at least three amino acids. The terms "protein" and "peptide" are used interchangeably. The term "peptide" can refer to an individual peptide or a collection of peptides. One or more amino acids in the disclosed imaging agents may be, for example, a carbohydrate group, a phosphate group, a pharmacophore, or a hydroxyl group. Addition of chemicals such as nesyl groups, isofarnesyl groups, sulfoxide groups, and fatty acid groups, They may be modified, such as with linkers for jugation, functionalization, or other modifications. In some embodiments, other modifications include the incorporation of D-amino acids, N- and C-terminal Other molecules conjugated to the end, such as fluorescent probes or poly(ethylene glycol), Conjugation of biomolecules, targeting ligands, etc., retroinversion, etc. may be included. Any modifications 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 a chelator, a radioactive Fluorescently labeled substrates, fluorescent dyes, photoacoustic reporting molecules, and Raman-active reporting molecules The compound is selected from the group consisting of:

[0029] In some embodiments of the imaging agent of the present disclosure, the reporting moiety is a chelator; The chelating agent is selected from the group consisting of: DOTAGA (1,4,7,10-tetrahydrofuran) azacyclododecane, 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[4.2.1.2]methyl ... chloro[5.5.2]tetradecane), DEPA (7-[2-(bis-carboxymethylamino) (no)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclo dodec-1-yl-acetic acid), 3p-C-DEPA(2-[(carboxymethyl)][5-(4- Nitrophenyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetramethyl tetraazacyclododecan-1-yl)pentan-2-yl)amino]acetic acid), TCMC(2 -(4-isothiocyanobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra tetra-(2-carbamonylmethyl)-cyclododecane), oxo-DO3A (1-oxa-4, 7,10-Triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,1 O-triacetic acid), p-NH2-Bn-oxo-DO3A (1-oxa-4,7,10-tetraazacin Chlododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A((1, 8--N,N'-bis-(carboxymethyl)-1,4,8,11-tetraazacyclotetradecane CAN), MM-TE2A, DM-TE2A, CB-TE2A (4,11-bis(carboxy) Methyl)-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-tetraazacyclotetra Decane-1,8-bis(methanephosphonic acid), TETA(1,4,8,11-tetraazacrylamide Chlotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclo Nonane-N,N',N''-triacetic acid), NODA (1,4,7-triazacyclononane-1 ,4-diacetate); NODAGA (1,4,7-triazacyclononane, 1-glutaric 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), T ACN-TM(-N,N',N',tris(2-mercaptoethyl)-1,4,7-triazolo[4- ... cyclononane), diamsal (1,8-diamino-3,6,10,13,16,19-hexahydroxybenzoate), Hexaazabicyclo(6,6,6)eicosane, 3,6,10,13,16,19-hexa Azabicyclo[6.6.6]eicosane-1,8-diamine, Sarar(1-N-(4- Aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6] ]eicosane-1,8-diamine), AmBaSar (4-((8-amino-3,6,10, 13,16,19-Hexaazabicyclo[6.6.6]icosan-1-ylamino)methyl ) benzoic acid), and BaBaSar.

[0030] In some embodiments, the peptide, linker, and reporter conjugate are They are prepared by conventional chemistry. See, for example, published international patent application to Pomper et al. WO / 2017 / 027870, triazole conjugated ureas, thioureas, carbamates Mate and PSMA-targeted imaging agents and and "reverse" carbamates for use therein, as well as U.S. Patent Application Publication No. 201403 41804 to Pomper et al., which was published on November 20, 2014. Homopolyvalent and heteropolyvalent inhibitors of prostate-specific membrane antigen (Pmsa) and their uses. No. 6,239,999, 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 L u, 186 Re, 188 Re, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 5 5 Co, 57 Co, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 15 3 Sm, 166 Ho, 152 Gd, 82 Rb, 89 Zr, and 166 The group consisting of Dy The radioactive metal further comprises a radioactive metal selected from the group consisting of:

[0033] In other embodiments of the imaging agents of the present disclosure, the reporting moiety is a radiolabeled substrate, The radiolabeled substrate is 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 radioisotope is selected from the group consisting of At In a more particular embodiment, the radiolabeled substrate comprises an F-labeled substrate. 18 F-labeled substrate are 2-fluoro-PABA, 3-fluoro-PABA, 2-fluoro-mannitol, and N-succinimidyl-4-fluorobenzoate. In embodiments, the substrate may be, for example, NOTA, NODA, or any other substrate known in the art. Based on the chelation of aluminum fluoride with any other suitable chelating agent, Al Using the F method 18 Labeled with F. See, for example, Liu S., et al. ., “One-step radiosynthesis of 18 F-AlF-NO TA-RGD2 for tumor angiogenisis PET imagi ng. Eur J Nucl Med Mol Imaging. 2011, 38 (9):1732-41; McBride WJ, et al., “A no vel method of 18 F radiolabeling for PET. J Nucl Med. 2009;50:991-998; McBride W. J, D'Souza CA, Sharkey RM, Sharkey RM, K aracay H, Rossi EA, Chang CH, Goldenber g 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, is selected from the group consisting of: carbocyanine, indocarbocyanine, oxaca Carbocyanine, carbocyanine, merocyanine, polymethine, coumarin, rhodamine , xanthene, fluorescein, boron-dipyrromethane (BODIPY) dyes, or Derivatives of BODIPY, such as BODIPY FL, BODIPY R6G, BODIPY TR, and BODIP Y TMR, BODIPY 581 / 591, BODIPY 630 / 650, and BO Cy5, Cy5.5, Cy7, including but not limited to DIPY 650 / 665 , VivoTag-680, VivoTag-S680, VivoTag-S750, Al exaFluor660, 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-dimethyl Aminophenyl)-2,4-pentadienylidene]-2,5-cyclohexadien-1-ylidene Ammonium Perchlorate), IRDye 800CW, IRDye 800RS, IR Dye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0035] In other embodiments of the imaging agents 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 particular embodiments, the fluorescent dye comprises: Indocyanine green (ICG), Alexa Fluor 750, Evans Bl ue, BHQ 3, QXL 680, IRDye 880CW, MMPSense 680, Selected from the group consisting of methylene blue, PPCy-C8, and Cypate-C18 . Wu et al., Int. J. Mol. Sci., 15, 23616 -23639 (2014).

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

[0037] In other embodiments of the imaging agents of the present disclosure, the reporting moiety is a Raman-active reporting moiety. molecule, and the Raman-active reporting molecule is a single-walled carbon nanotube (SWNT). and surface-enhanced Raman scattering (SERS) agents. The SERS agent is a metal (e.g., gold or silver) labeled with a Raman-active reporter molecule. In more particular embodiments, the Raman-active reporter molecule comprises a fluorescent dye. In certain embodiments, the fluorescent dyes include Cy3, Cy5, rhodamine, and chalcogenides. The dye is selected from the group consisting of pyrylium 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 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 1, 2, is an integer selected from the group consisting of 3, 4, 5, 6, 7, and 8; and The 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 are each independently 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 0 to 5 R A Y and W3 may be substituted with Independently, -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 A is a halogen, hydroxyl, , amino, cyano, nitro, CO2H, optionally substituted alkyl, optionally substituted s cycloalkyl, optionally substituted heterocycle, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted mono- or di-alkynyl alkylamino, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted mono- or dialkylcarboxy amide, optionally substituted aryl, or optionally substituted heteroaryl; R B Each occurrence of is an optionally substituted alkyl, an optionally substituted alkoxy ... optionally substituted mono- or dialkylamino, optionally substituted alkylthio, optionally substituted aryl, or optionally substituted heteroaryl); or (c) Amino acid linker.

[0039] In certain embodiments, the imaging agent comprises a compound of formula (I), formula (II), and formula (III): is a compound selected from the group: [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 site; and The anchor, when present, is a pen containing an imaging agent of formula (I), formula (II), or formula (III). (attached to the primary amine group of the peptide).

[0040] In certain embodiments, the linker, when present, is 13 Ornithine (Orn) is attached to a primary amine group. In certain embodiments, the reporting moiety is D In a more particular embodiment, the DOTAGA chelator 6 4 Further includes Cu radiometal.

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

[0042] Those skilled in the art, upon consideration of the presently disclosed subject matter, will recognize key compounds suitable for use with the imaging agents disclosed herein. It will be appreciated that various rate agent / radiometal ion combinations are suitable. Representative chelating agents are known in the art. Non-limiting examples include certain The chelators and linkers are described in U.S. Patent Application Publication Nos. 2015 / 0246144 and 2015 / 0246144. and US Pat. No. 2015 / 0104387, each of which is incorporated by reference in its entirety. and is incorporated herein by reference.

[0043] In some embodiments, the imaging agent is PD-L1 can be detected ex vivo or ex vivo. can detect PD-L1 in vivo. PD-L1 is expressed by various tumors. Its overexpression is thought to promote tumor proliferation as an adaptive mechanism in response to tumor-infiltrating cytotoxic T cells. PD-L1 is induced in tumor cells (Topalian et al., 2016). may contain modifications and / or mutations that can still be detected by the imaging agents of the present disclosure. It will be appreciated that, to the extent that such modifications are possible, they may still be applicable to the methods of the present disclosure.

[0044] In some embodiments, PD-L1 and its ligand, programmed cell death protein IC of the imaging agents of the present disclosure that inhibit interaction with protein PD-1 50 is approximately 100nM In some embodiments, the IC 50 is less than 100 nM, other In some embodiments, less than 10 nM; in other embodiments, less than 8 nM; in other embodiments, less than 5 nM; In some embodiments, it is less than 4 nm, and in other embodiments, it is less than 3 nM.

[0045] The term "binding affinity" refers to how strongly two or more compounds associate with each other in a non-covalent manner. Binding affinity can be described qualitatively (such as "strong," "weak," or "high"), or "low") or quantitatively (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 is directed to immune checkpoint transcription factors such as PD-L1. In some embodiments, the presently disclosed subject matter provides a method for detecting a protein comprising: Diseases, disorders, or conditions that result in overexpression of PD-L1, such as cancer, inflammation, and infection A method for detecting the

[0047] In some embodiments, the presently disclosed subject matter provides a method for producing a programmed death ligand, comprising the steps of: Imaging methods for detecting programmed death ligand 1 (PD-L1): (a) A peptide with binding specificity for PD-L1 (PD-L1) and a reporting domain An imaging agent comprising a conjugate, and optionally a linker, as described immediately above, If present, the carrier connects the peptide to the reporting moiety, and if present, the linker If not, the reporting moiety is attached to the peptide via the primary amine of an amino acid of the peptide. (b) providing an effective amount of an imaging agent directly attached to one or more microspheres; (c) contacting the cell or tissue with an imaging agent; and (c) generating an image to detect PD-L1. Process.

[0048] As used herein, the term "imaging" or "creating an image" means Visualize detectable compounds by measuring the energy emitted by the compound In some embodiments, "imaging" refers to the use of any imaging technique. The term "energy release" refers to the measurement of the energy released from a compound after localization of the compound following administration. by any imaging technique that visualizes a detectable compound after administration to a subject. In some embodiments, the imaging technique involves detection from outside the subject. In some embodiments, the image comprises administering to the subject a compound capable of: It is produced by differences in the spatial distribution of the contrast agent that accumulates in different parts of the subject. In an embodiment, the administration of the contrast agent is by injection.

[0049] The term "imaging agent" refers to a substance that can be imaged, for example, by positron emission tomography (PET). As used herein, "positron emitting" is intended to include compounds that are capable of "PET" stands for "positron emission tomography imaging" and refers to all positron emission tomography imaging. systems or equivalent, and all equipment capable of positron emission tomography imaging. The methods of the presently disclosed subject matter may be used with any such device, or with a PET device or equivalent. It can be implemented using variations of the same or in conjunction with any known PET methodology. See, for example, U.S. Patent No. 6,151,377, each of which is incorporated herein by reference. 7;6,072,177;5,900,636;5,608,221;5,532,48 9;5,272,343;5,103,098. Examples include microPET (Corcorde Microsystems) do.

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

[0051] In some embodiments, imaging is performed using a detection system to detect a subject or The entire patient or specific areas of the subject or patient may be scanned to detect the signal. The detected signal is then converted into an image. The resulting image can be used for, e.g., medical advice. The results should be interpreted by a skilled observer. Generally, the results are obtained approximately 1 minute to 1 hour after administration of the contrast agent. Imaging is performed at 48 hours. The exact timing of imaging is within the skill of the art. As will be readily apparent, this will depend on factors such as the clearance rate of the administered compound. Imaging time frames 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., 15 minutes. Between 30 minutes and 45 minutes, between 45 minutes and 60 minutes, between 60 minutes and 90 minutes In some embodiments, the detection of PD-L1 is performed between 60 and 120 minutes. is performed no sooner than about 60 minutes after administering the imaging agent to the subject. In this study, imaging was performed 24 hours after injection of the Zr-89 labeled peptide. In some embodiments, imaging may involve the detection of I-124 labeled peptides. This may be done 24 hours after injection.

[0052] Once an image is obtained, one skilled in the art can determine the location of the compound. Thus, one of skill in the art can easily determine whether a condition, such as, for example, an infection, inflammation, or cancer, is present and how the condition affects the patient. The extent of the condition or the effectiveness of a treatment the subject is receiving can be determined.

[0053] In some embodiments, contacting the cells or tissue with the imaging agent is performed in vitro, i.e. "Contacting" refers to contacting a subject of the present disclosure in vivo or ex vivo. At least one imaging agent is in physical contact with at least one cell or tissue. It therefore means any effect that is produced by the combination of at least one contrast agent and at least a cell or cells in an amount sufficient to bring the cell or cells into contact with at least one cell or tissue. ) or exposing the tissue(s) to a contrast agent. In embodiments, the method involves placing the contrast agent and cells or tissue in a controlled environment, such as a culture dish or tube. by introducing and preferably mixing the tissue in vitro or ex vivo In some embodiments, the method can be performed in vivo. In this case, the contacting may involve contacting at least one cell or tissue of the subject with the Exposing the subject to at least one imaging agent, e.g., administering the imaging agent via any suitable route. In some embodiments, the term "administration" refers to administration to a subject via a cell or The contacting of the tissue with the contrast agent occurs within the subject.

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

[0055] In many of these embodiments, the subject to be diagnosed or treated by the methods of the present disclosure is The subject is preferably a human subject, although the methods described herein may be used in conjunction with the term "subject." It should be understood that the invention is effective with respect to all vertebrate species intended to be included therein. Thus, a "subject" is a person who is being treated medically, such as for the diagnosis or treatment of an existing disease, disorder, or condition. human subjects for clinical purposes, or for medical, veterinary, or developmental purposes Suitable animal subjects include primates, e.g., humans. This includes monkeys, apes, gibbons, chimpanzees, orangutans, macaques, etc. Mammals, including but not limited to: cattle, such as cows, cattle, etc.; ovine, such as sheep, etc.; goats , such as goats; swine, such as pigs and hogs; equids, such as horses, donkeys, zebras cats, including wild and domestic cats; canines, including dogs; lagomorphs, rabbits, including rabbits, hares, etc.; rodents, including mice, rats, guinea pigs, etc. Animals are genetically In some embodiments, the subject may be a fetus, a neonate, an infant, a juvenile, and humans, including, but not limited to, adult subjects. 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. The subject may be an animal disease model (e.g., an experimental rat or mouse). In some embodiments, the subject is a human, a rat, a mouse, a cat, a dog, a cow, a cat ... It may be a macaque, sheep, cow, monkey, bird, or amphibian.

[0056] In general, the imaging agents of the present disclosure can be administered to treat diseases, disorders, or may be administered to a subject for the detection of a condition: orally, nasally, transmucosally, intraocularly, intrarectally, Intravenous, intramuscular, subcutaneous, and intramedullary injections, including vaginal or parenteral, as well as intrathecal and direct brain administration Intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or It can be administered intraocularly, intracapsularly, topically, by powder, ointment or drops (including eye drops), orally. Intracavitary and sublingual administration, transdermal administration, via inhalation spray, or other methods known in the art. Other modes of delivery.

[0057] As used herein, "systemic administration," "systemic administration," "peripheral administration," and "peripheral administration" are used interchangeably. The term "administered" refers to the fact that they enter the system of a subject or patient and are therefore metabolized and otherwise affected. This refers to administration of a composition such that it undergoes a similar process, e.g., subcutaneous or intravenous administration. do.

[0058] As used herein, the terms "parenteral administration" and "parenteral administration" are generally used with caution. means modes of administration other than enteral and topical administration, including intravenous, intramuscular, intraarterial, Intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, including, but not limited to, subthecal, subarachnoid, intraspinal and intrasternal injections and infusions stomach.

[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 a cell or cells that exhibit overexpression of PD-L1 protein. The term "non-target" refers to cells or tissues that do not exhibit overexpression of the PD-L1 protein. Refers to an organization.

[0060] In some embodiments, the imaging method is used to detect cancer. "Cancer" in a body or patient is defined as the development of cells that have characteristics typical of cancer-causing cells. Presence of, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, anti-apoptotic Significantly increased cis activity, rapid growth and proliferation rates, and certain characteristic morphologies and cellular markers In some circumstances, cancer cells take the form of tumors; such cells grow locally within an animal. They can be present locally or circulate in the bloodstream as independent cells, e.g., leukemia cells. As used herein, cancer includes blastoma, carcinoma, glioma, leukemia, lymphoma, melanoma, bone marrow tumor, and the like. Newly diagnosed or recurrent myeloma, including but not limited to myeloma, and sarcoma As used herein, cancer includes head cancer, neck cancer, head and neck cancer, lung cancer, Breast cancer including ripple-negative breast cancer, prostate cancer, colon cancer, esophageal cancer, stomach cancer, white blood cell Disease / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary tract cancer, pancreatic cancer, gastrointestinal cancer, Including, but not limited to, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, brain tumors, and adenomas In some aspects, the cancer comprises stage 0 cancer. The cancer comprises stage I cancer. In some embodiments, the cancer comprises stage II cancer. In some embodiments, the cancer comprises stage III cancer. In some embodiments, the cancer is refractory and / or metastatic, including stage IV cancer. be.

[0061] As used herein, a "tumor" refers to any neoplastic cell, whether malignant or benign. refers to the growth and proliferation of cells and tissues, as well as all precancerous and cancerous cells and tissues. As used herein, a "solid tumor" is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors include, but are not limited to, tumors of the brain, colon, breast, prostate, liver, kidney, lung, esophagus, May be present in the head and neck, ovaries, cervix, stomach, colon, rectum, bladder, uterus, testes, and pancreas In some embodiments, the imaging method is used to detect solid tumors. In yet another embodiment, the imaging method is used to detect metastatic cancer. .

[0062] In some embodiments, the imaging method is used to detect an infectious disease. Infectious diseases, such as fungal or bacterial infections, are suitable for detection using the subject matter of the present disclosure. As used herein, the term "infection" refers to an organism that causes disease. invasion of the body tissues of host organisms by bacteria, their proliferation, and the proliferation of these organisms and the products they produce. It refers to the response of host tissue to the toxin produced by the bacteria. severe abdominal infections such as peritonitis, pancreatitis, gallbladder empyema and pleural empyema, as well as osteomyelitis Bone infections include, but are not limited to, sepsis, septicemia and septic shock. Detection of infections due to or after the use of immunosuppressants, cancer chemotherapy, radiation, and contamination Infusion, hemorrhagic shock, ischemia, trauma, cancer, immunodeficiency, viral infection, and diabetes Examples of microbial infections, such as bacterial and / or fungal infections, include Mycobacterium tuberculosis, , Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Serratia marcescens Staphylococcus aureus, including Pseudomonas aeruginosa, Staphylococcus aureus, and coagulase-negative staphylococci Enterococcus spp., Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides spp., Acinetobacter spp. Infections include, but are not limited to, infections caused by Tobacter, Helicobacter, and Candida. Infections caused by resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA) and and vancomycin-resistant Enterococcus faecium (VRE). 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 is hepatitis A, hepatitis B, It may be hepatitis C, and / or human immunodeficiency virus.

[0063] In some embodiments, imaging methods are used to detect inflammation. Examples of disorders related to asthma include autoimmune and autoinflammatory diseases, celiac disease, and eczema. Vaccinitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis media, pelvic Inflammatory diseases, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection Reactions, lupus including systemic lupus erythematosus, and vasculitis In some embodiments, the inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus. This is caused by

[0064] PD-L1 is its receptor found on activated T cells, B cells, and myeloid cells. It binds to PD-1, which is involved in the activation or inhibition of PD-L1. The imaging agents of the present disclosure are useful for detecting immune cells such as T cells, B cells, and myeloid cells. In some embodiments, the imaging agents of the present disclosure can be used to detect immune cells in tumors. In some embodiments, the imaging agent of the present disclosure detects the distribution of immune cells in a subject. In some embodiments, the imaging method detects the presence of HIV in infected cells. In some embodiments, the imaging method is used to detect immune cell responses. The method is used to detect immune cell responses in inflammatory cells.

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

[0066] III. Kits Containing Contrast Agents In some embodiments, as described above, the presently disclosed subject matter comprises a method for treating a death-associated cell death syndrome (DDS) by inhibiting the activation of a cell death receptor (CDR) in a mammalian cell. The present invention provides a kit for detecting programmed death-ligand 1 (PD-L1), L1), a conjugate of a peptide having binding specificity for the reporting moiety; and optionally a linker, wherein the linker, if present, is a peptide and the reporting moiety, and if no linker is present, the reporting moiety includes an imaging agent directly attached to a 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 at least one The imaging agent typically binds at least one PD-L1 receptor. Provided in the kit in an amount sufficient for at least one detection in an individual subject or patient. The kit may include other components necessary to practice at least one embodiment of the disclosed method. Some or all of the reagents and supplies may also be included.

[0068] In its simplest form, a kit of the presently disclosed subject matter comprises at least one In some embodiments, the kit comprises a plurality of containers, Each container contains at least one imaging agent or one or more embodiments of the disclosed method. The composition may contain other substances useful for

[0069] The container may contain a composition of the present disclosure or other material useful for practicing the methods of the present disclosure. The container may be any material suitable for the purpose. Thus, the container may be a vial or an ampoule. It may be made of any suitable material such as glass, plastic, metal, or paper or paper products. In an embodiment, it can be made from a material such as a stopper, a stopper and Sealed with a crimp seal or plastic or metal cap The container is a glass or plastic ampoule or vial. The amount of contrast agent included may vary based on a number of parameters relevant to the subject matter of this disclosure. These can be selected by one skilled in the art without experimentation.

[0070] In embodiments, the container is a component of a larger unit that typically contains packaging material. The kits of the present disclosure are provided in suitable packaging (hereinafter referred to as kits). and instructions, and / or other information regarding use of the composition. The box is constructed of sturdy materials such as cardboard and plastic and contains no instructions or other information. The kit may include multiple containers containing the compositions of the present invention. In such a kit, each container is the same size as each other container and contains the same amount of composition. Alternatively, different containers may be different sizes and / or contain different amounts of The container size and contents may include a composition or a composition having different ingredients. Many different configurations of are contemplated by the present invention, and therefore all permutations are included herein. It will be readily apparent that the invention does not necessarily have to be specifically listed in the book.

[0071] Although specific terms are employed herein, they are used in a generic and descriptive sense only. Unless otherwise defined, the terms used herein are used in their entirety and not for purposes of limitation. All technical and scientific terms used herein are to be understood as meaning the term "technical" or "scientific" as used herein to refer to the art to which the subject matter described herein belongs. It has the same meaning as commonly understood by one of ordinary skill in the art.

[0072] Following long-standing patent law practice, the terms "a," "an," and "the" are used to refer to the parts of a patent claim. When used in this application, including ranges, it refers to "one or more." For example, unless the context clearly indicates otherwise (e.g., multiple subjects), " includes a plurality of subjects.

[0073] Throughout this specification and claims, the terms "comprise," "include," and "comprise" " is used in a non-exclusive sense unless the context otherwise requires. The term "contains" and its grammatical variations mean that the enumeration of items in a list is The terms "item" and "item" are non-exclusive and do not exclude other similar items that may be substituted or added to the item. It is intended to be.

[0074] For purposes of this specification and the appended claims, unless otherwise indicated, amounts, Size, dimensions, proportion, shape, composition, parameter, percentage, parameter, amount, characteristic, arrangement Other numerical values used in this specification and claims are intended to be used only if the term "about" is used to refer to that value, In all cases, the term "about" is used, even if not expressly appearing with any quantity or range. Therefore, unless indicated to the contrary, the following statements should be understood to be modified in their entirety. The numerical parameters set forth in the specification and attached claims are not precise and should not be construed as limiting the scope of the invention. It does not have to be, but it can be approximate and / or larger or smaller, tolerances, conversions coefficients, rounding, measurement errors, etc., as well as the results sought to be obtained by the subject matter of this disclosure. The values reflect other factors known to those skilled in the art that depend on the desired properties of the material. The term "about" when referring to a particular amount means, in some embodiments, ±1 00%, in some embodiments ±50%, in some embodiments ±20%, in some embodiments In some embodiments, ±10%, in some embodiments ±5%, and in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1% variation; Such variations may be suitable for practicing the disclosed methods or employing the disclosed compositions. It can mean inclusion where appropriate.

[0075] Additionally, the term "about" when used in connection with one or more numbers or numerical ranges. When a range is used, it should be understood to refer to all such numbers, including all numbers within the range, and Modify the range by extending the bounds above and below the specified numeric value. A list of all numbers included within that range, e.g., all decimal points included within that range. the whole integers (e.g., the enumeration 1-5 includes 1, 2, 3, 4, and 5, and their decimals) , e.g., 1.5, 2.25, 3.75, 4.1, etc.) and any value within that range. Contains a range of: [Example]

[0076] The following examples are provided to provide guidance to those of skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of this disclosure and the general state of the art, The following examples are intended to be illustrative only, and numerous variations, modifications, and alternatives are possible within the scope of the present disclosure. Those skilled in the art will understand that the following may be adopted without departing from the scope of the present invention. The synthetic descriptions and specific examples are intended for illustrative purposes only and are not to be construed as limiting the scope of the invention. Therefore, nothing in this disclosure should be construed as limiting in any way in which the compounds of this disclosure may be prepared. isn't it.

[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) promotes active immune infiltrates Immunity via binding to the programmed cell death protein 1 (PD-1) receptor expressed by causes immunosuppression by inactivating the immune infiltrate (Okazaki et al., 2007, and PD-L1 expression on tumor cells and in the TME is associated with a decline in tumor size and progression in patients with advanced or advanced PD-L1 disease. It is considered a potential biomarker for stratification and therapeutic monitoring of Herbst et al., 2014). A supplemental diagnostic test based on PD-L1 IHC is available in the US Food and Drug Administration. Recently approved by the National Institute of Health, the PD-L1 receptor is suitable for in vivo imaging. suggest that it may be a target (Roach et al., 2016).

[0078] Currently, immunohistochemistry (IHC) detection is used for therapeutic monitoring of PD-L1 / PD-1 targeted therapy. Although it is the most studied predictive biomarker for PD, this approach and The available FDA-approved diagnostic IHC test for -L1 has significant limitations, ch et al., 2016; Mansfield and Dong, 2016; and Phil ips et al., 2015, inconsistencies in antigen positivity definitions, discrepancies in detected antibodies, and lack of agreement between assays adequacy, as well as intra- and inter-tumor heterogeneity that compromises accuracy and reliability, and therefore treatment and the tissue samples obtained by biopsy for testing. Samples are usually very limited and targetable oncogenic mutations in other pathways ( For example, epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase, and DNA repair genes ), which may be necessary for molecular profiling to identify existing These valuable samples are characterized by PD-L1 expression and are sensitive or resistant to treatment. This often makes it impractical to perform multiple PD-L1 assessments for reliable delineation of It allows non-invasive assessment of PD-L1 expression levels, kinetics, and distribution, and is effective within 60 minutes of administration. Novel PET contrast agents that perform imaging within the standard clinical workflow of PD -overcomes the shortcomings of available (IHC-based) methods for assessing L1 expression status Deaf.

[0079] The dynamic nature of the tumor immune microenvironment necessitates the use of PET tracers that allow rapid assessment of the TME. In this regard, we provide a rationale for the development of low molecular weight peptide-based PET trays. Serum is an attractive candidate for clinical application due to its fast clearance and ease of synthesis. Somatostatin is a desirable candidate (Reubi et al., 2008; Sun et al., 2016). Peptide-based PET targeting receptors and chemokine receptor 4 (CXCR4) The tracer produces a high target-to-non-target ratio in patients (Herrmann et al., 2016 ;Gourni et al., 2011).

[0080] Recently, peptides that specifically bind to PD-L1 have been reported (see below: 201 Miller et al., International PCT Patent Application Publication No. WO201, published March 17, 2016 6039749, Macrocyclic Inhibitors of the PD -1 / PD-L1 and CD80 (B7-1) / PD-L1 Protein / Pro tein Interactions;Published June 23, 2016, Mapel Li et al., International PCT Patent Application Publication No. WO2016 / 100285, Immunomod ulators; International PCT patent application publication by Sun et al., published June 23, 2016 Opening number WO2016 / 100608, Immunomodulators; 2016 8 Miller et al., International PCT Patent Application Publication No. WO2016 / 1 26646, Immunomodulators, each of which is incorporated herein in its entirety. however, their ability to detect PD-L1 expression in vivo is uncertain. These PD-L1 binding peptides rapidly increase PD-L1 expression in tumors. It was hypothesized that it could be detected with high specificity and accuracy. The reported peptides with a single primary amine are most suitable for ligation. A peptide, WL12, was selected from the library and its binding mode to PD-L1 was evaluated. TAGA chelating agent 64 For radiolabeling with Cu, conjugate with WL12 and 64 Cu WL12 (Eisenwiener et al., 2000) and a peptide against PD-L1. We evaluated the binding affinity of tide derivatives and compared their binding affinity in cell lines with variable PD-L1 expression. 64 Cu ]WL12 in vitro uptake was evaluated. 64 Cu]WL 12 demonstrated the ability to detect PD-L1 expression by PET imaging in vivo. Chinese hamster ovary (CHO) mice with constitutive human PD-L1 expression (hPD-L1) ) tumor and isogenic negative control tumor (CHO)-bearing NSG mice. [ 64 The tissue distribution and target specificity of [Cu]WL12 were investigated using ex vivo biodistribution and This was confirmed by a blocking test.

[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 to D-L1, we docked WL12 instead of PD-1. The co-crystal structure of human PD-L1 bound to PD-1 (PDB ID: 4ZQK) (Za Considering the structural complexity of the macrocycle WL12, the present inventors First, a conformational search was performed to identify the PD-1 binding site on PD-L1 using Glide. The conformational isomers were docked (Friesner et al., 2004; Halgren et al., 200 4) WL12 has 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 binding between the two. The two base domains of PD-1 that form the binding interface with PD-L1 reveal similarities in their modes of action. The WL12 L-leucine residues overlap with the WL12 pseudochain (Fig. 1C). It is inserted into the same small hydrophobic pocket as e134 and is one of two norleucine residues aligns with Ile126 of PD-1. In addition to these hydrophobic interactions, numerous hydrogen A bond exists between WL12 and PD-L1. The amide forms a hydrogen bond with Tyr123, and the glycinamide forms a hydrogen bond with the backbone of Gly120. The serine hydroxyl interacts with Gln66, and the ornithine residue is exposed. Without wishing to be bound by any one particular theory, Although not desirable, this allows for the conjugation of suitable labels by amine coupling methods. These results suggest that the antibody does not interfere with the binding of WL12 to PD-L1.

[0082] 1.2.2:[ 64 Cu]WL12 inhibits PD-L1-specific cell uptake in vitro Indicates congestion. 13 Ornithine (Orn) primary amine is used to conjugate DOTAGA This is then used to gate the non-radioactive C 2+ An analogue (WL12-Cu) was prepared, 6 4 The resulting WL12D and the corresponding WL12-Cu were radiolabeled with Cu. and characterized by mass spectrometry (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, and Figure 7) was evaluated in vitro. WL inhibits the interaction of PD-L1 with PD-1. The half-maximal inhibitory concentration (IC) of 12 and its derivatives 50 ) to evaluate the fluorescence resonance energy We optimized a previously described in vitro assay that relies on energy transfer (Woodard et al. , 2014). WL12, WL12D, and WL12-Cu were 22 and 22, respectively. 23, and an IC of 2.9 nM 50 values were observed (Figures 8A, 9, 10 and below). Table 1). These data are 13 Orn side chain was modified with DOTAGA and Cu 2+ Replying to @Kira This indicates that WL12 maintains high binding affinity for PD-L1 even after immunohistochemistry. There are.

[0083] [Table 1]

[0084] To demonstrate PD-L1 specificity and cellular uptake, 64 Cu]WL12 Produced with specific radioactivity (1.9±0.1mCi / μg) and radiochemical purity (>95%) (Figures 11 and 12). 64 hPD-L incubated with [Cu]WL12 for 1 hour 1 cells showed >50% uptake of the incubated dose compared to negative control CHO cells. hPD-L1 cells were then transfected with 1000 mAbs of ... 64 Cu]WL12 alone or in the presence of a blocking dose of 1 μM WL12. The binding specificity was tested by incubating the 64 Cu]WL12> A 95% reduction in PD-L1 activity was observed in the presence of the peptide. 64 Cu]WL12 The results showed that the combination of PD-L1 and PD-L1 expression was specific (Figure 8C). 64 Cu WL12 performance compared with two triple-negative tumors with high and low PD-L1 expression, respectively. and in the transverse myeloma (TNBC) cell lines MDAMB231 and SUM149. (Figure 8B). Radioactivity in MDAMB231 cells compared to SUM149 cells. The two-fold higher uptake of PD-L1 64 Further confirming the specificity of Cu]WL12 Flow cytometry analysis of PD-L1 expression was performed in the following order: The mean fluorescence intensity values were: hPD-L1 > MDAMB231 > SUM149 > CHO. This correlated with the uptake of radioactivity (r=0.9977, Figures 13 and 14). And these results are 64 Cu]WL12 is dependent on PD-L1 expression in vitro It has been demonstrated that the compound binds to cancer cells.

[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 demonstrated the presence of hPD-L1 in tumors. 64 Cu]WL The increased uptake in hPD-L1 tumors was observed within 10 minutes. This was observed as early as 24 hours after injection (Figure 15A and Figure 16), and PD-L1 Expression was confirmed by IHC (Figure 15B). In addition to tumors, expression was also high in kidney and liver. To confirm the PET imaging observations, a biodistribution study was performed. 64Cu] 1 and 2 hours after injection of WL12 (n = 3 and n = 5, respectively). Given the rapid uptake observed in PD-L1-positive tumors, The biodistribution of 18F was considered to be more beneficial for the development of 18F-labeled analogues. Consistent with the immunization study, hPD-L1 tumors expressed 14.9 ± 0.8 injected dose / g (%I) at 1 hour. Radioactivity uptake was expressed as a percentage of the (D / g) value. In contrast, control CHO tumor uptake was The uptake in the kidney and liver was also relatively high. The uptake values were 34.4±3.1 and 24.2±2.5%ID / g, respectively. The tumor-to-muscle and tumor-to-blood ratios of hPD-L1 tumors were 25.6 ± 1.9 and 26.2 ± 1.9, respectively. and 4.7±1.2, and 64 Cu]WL12 shows high signal in PD-L1 specific images This was consistent with the performance provided by the signal-to-noise ratio (Figures 15A and 15B).

[0086] Biodistribution studies performed after 2 hours showed a decrease in radioactivity in the kidney, liver, and tumor. The results showed similar profiles with a trend towards a 100% specificity (Figure 17). In order to 64 Cu]WL12 was administered simultaneously with excess WL12 (50 μg, 2 mg / kg). The PD-L1 tumors were injected and biodistribution was performed at 2 hours. A 75% reduction (P<0.0001) was observed, with no significant difference observed in control CHO tumors. There was no significant difference in radioactivity uptake in other tissues. No increase in hepatic uptake was observed. 64 Often observed with Cu-based contrast agents The tendency for Cu to be released from chelating agents (Anderson et al., 2009) 2+ Dissociation of and Subsequent transchelation to plasma proteins such as albumin and ceruloplasmin This may be due to silicification (Smith-Jones et al., 1991; Wadas et al., 2007; and Boswell et al., 2004). Increased renal uptake may contribute to renal clearance of peptides. These tumors are known to express PD-L1 and are involved in the uptake of radiolabeled antibodies. In the spleen, thymus, and brown fat, tissues that have been reported to show increased , low uptake was observed (Chatterjee et al., 2016; Hettich et al., 2017). 016; and Josefsson et al., 2016), [ 64 Cu]WL12 is a mouse P This suggests that the antibody has very low or no affinity for human PD-L1. [ 64 Cu] Further supporting WL12 specificity, except for the kidney, No significant differences in uptake were observed between the control and blocking dose groups in these tissues. Imaging and biodistribution studies were conducted jointly by [ 64 Cu]WL12 is a human PD-L We demonstrate rapid and specific binding to 1.

[0087] 1.2.4: CD results. Evaluating the secondary structure of WL12 in aqueous and membrane-mimetic solutions. To determine the solubility of the solubility-rich solubility, CD spectroscopy was performed with a combination of water, DPC, and SDS. As shown, Trp residues are present in the CD spectrum of the WL12 peptide in the 220–240 nm region. In surfactant-free solutions, the minimum is at about 220 nm and the peak is at about 2 A positive shoulder was observed at 30 nm. Addition of surfactant reduced both bands. The latter band is slightly red-shifted and the latter band is increased in intensity. Both Trp chromophores are in close proximity, forming a single adsorption As a result, their excited states interact, and the excited state of the dimer system is This phenomenon, called the excitonic effect, occurs when the excited state This leads to splitting into two components, one of which arises from the in-phase combination of two monomer excitations. and the other arises from heterophase bonding (Grishina 1994, and Kelly 2000).

[0088] The CD spectrum of a disordered peptide is typically characterized by a single band below 200 nm. The α-helix was characterized by two negative bands at 208 and 222 nm. The β-sheet structure usually shows one positive band at 192 nm and one negative band at 217 nm. The positive band at 195 nm is shown. Therefore, the C of the WL12 peptide A strong negative band at -205 nm and a strong positive band at -190 nm on the D spectrum The reactive bands may suggest a mixture of random coil and more ordered structures. Deconvolution of the CD spectra revealed a high β-sheet content (~ 40%). Nevertheless, the Trp coloration in the far-UV CD spectrum of WL12 The strong contribution of the nucleotides from the nucleotide sequence affects the accuracy of quantitative analysis of secondary structure content, and the results should be interpreted with caution. It should be.

[0089] 1.3 Summary: In summary, rapid tumor PD-L1 detection and PD-L1 selectivity are highly Specific PD-L1 binding peptide [ 64 Cu]WL12 in vitro and This was demonstrated in vivo by PET. 64Cu]WL12 pharmacokinetics and Biodistribution showed that PD-L1 detection was detected within 60 minutes of radiotracer administration in patients with imaging This suggests that it is feasible to fit the device into the standard clinical workflow for Rapid and non-invasive detection of PD-L1 expression across all malignancies may be useful in assessing immune regulation. It offers an unprecedented opportunity to stratify patients for therapy.

[0090] 1.4 Materials and Methods 1.4.1 Materials: PD-L1 binding peptide, WL12, was obtained from CPC Sc with a purity of >95%. Custom synthesized by ientific (Sunnyvale, CA). All other chemicals were purchased from Sigma-Aldrich or Fischer unless specified. It was purchased from Her Scientific. 2,2',2'-(10-(2,6-dithiothiazolinone) xotetrahydro- 2H-pyran-3-yl)-1,4,7,10-tetraazacyclododeca (DOTAGA anhydride) and [ 64 Cu]Cl2, CheMatech Macrocycle Design Technology gies (catalog no. C109; Dijon, France) and the University of Wisconsin Unless otherwise specified, all cell culture-related reagents were purchased from Invitrogen. We purchased polyclonal anti-human IgG-Eu3+ Cryptate (catalog number 61HF) CKLA) and XL665-conjugated mouse monoclonal anti-6 histidine antibody ( Catalog number 61HISXLA) was purchased from Cisbio Assays (Bedford, MA). Recombinant human PD-1 Fc chimeric protein (Catalog) was purchased from the company Eppendorf (S.C.). (B7-H1)-His-tag (B7-H1) and recombinant human PD-L1 (B7-H1) The protein (catalog number 9049-B7) was purchased from R&D Systems (Minneapolis, MN). , Minnesota).

[0091] 1.4.2 Docking test: 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 Maestro Protein Preparation. Wizard in Maestro (Schrodinger Release 2 016-2: Maestro, version 10.6, Schrodinge r, LLC, New York, NY, 2016) was first prepared using (S Astry et al., 2013), which involves assigning bond orders and formal charges, and attaching hydrogen atoms. The hydrogen bond network within the protein is optimized. (reorientation of thiol and hydroxyl groups, sanitization of Asn, Gln, and His side chains) (including prediction of the protonation states of His, Asp, and Glu), followed by A simple minimization is performed using Prime Conform. The PD-1 structure has been removed. ational Search (Schrodinger Release 2016 -2: Prime, version 4.4, Schrodinger, LLC , New York, NY, 2016) to perform conformational analysis of the WL12 structure. A coordinate search was performed. The 100 lowest energy conformers were selected for the docking experiment. Glide (Schrod) was run using default settings and the input ring conformation. inger Release 2016-2: Glide, version 7.1 , Schrodinger, LLC, New York, NY, 2016). Docking was performed (Friesner et al., 2004; Halgren et al., 2004). The software used for these calculations was created by SBGrid (Mori n et al., 2013).

[0092] 1.4.3 Circular dichroism (CD) measurements: in surfactant-free aqueous solution and in dodecyl In aqueous micellar solution of phosphatidylcholine (DPC) and sodium dodecyl sulfate (SDS) The CD spectra of the peptide in mixed DPC:SDS micelles with a molar ratio of 5:1 were measured by Ja All data were obtained using a Jasco J-815 spectropolarimeter (Jasco, Easton, MD). The measurements were carried out at 25°C using a 0.15 mg / mL peptide solution. The measurements were performed over a range of 260 nm and in triplicate to increase the signal-to-noise ratio. The final spectra were corrected by background subtraction and the mean residue molar ellipticity, MRME ( degrees x cm 2 ×dmol -1 The secondary structure content was analyzed as CO It was calculated from the spectra using the NTIN 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 3.7 mg of anhydrous DOTAGA (0.5 7.51 μmol in mL of DMF) and 20 μL of diisopropylethylamine (DI The reaction mixture was stirred at room temperature for 2 hours, and the product was purified by reversed-phase high-performance liquid chromatography. Agi was analyzed using a RP-HPLC system (Varian ProStar). Lent Technology 1260 Infinity Photodiode Array Detector with a detector (Agilent Technologies, Wilmington, DE). A semi-preparative C-18 Luna column (5 mm, 10 × 250 mm, Phenomenex, Tono) was used. (France, CA) and 98% HO (0.1% TFA) and 2% MeOH ( Starting with 0.1% TFA, reach 100% MeOH in 60 min at a flow rate of 4 mL / min The desired WL12D was collected at 44.5 min and evaporated. Dissolved in deionized water and lyophilized to give 3.1 mg (1.3 μmol) of product as a white solid. The resulting conjugate was obtained as a powder (yield: 82.9%, Figure 2). The samples were solubilized in 50% (v / v) HO-MeOH containing acid and analyzed by electrospray ionization mass spectrometry. Analysis method (ESI MS, Esquire 3000 Plus spectrometer, Bruker Da The results were analyzed by fluorochemical analysis (Waltonics, Billerica, MA) (Figure 4). Formula: C91H128N22O20S2. Observation 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+ Preparation of the complex: 1.5 mg of WL12D (0.64 μm ol) was dissolved in 200 μL of sodium acetate (0.1 M, adjusted to pH 4.5 with glacial acetic acid). The mixture was dissolved, and 55 μL of a 0.02 M aqueous solution of CuCl2 (1.1 μmol) was added. The resulting reaction mixture was incubated at 65°C for 30 min and then incubated as described for WL12D. The resulting pale blue powder was purified by RP-HPLC (Figure 5), freeze-dried, and analyzed by ESI MS. (Figure 6). Then, as a radiolabeled standard, and PD-L1 and P WL12-Cu as a standard for D-1 competitive binding assay 2+ Complexes were analyzed under RP-HPLC conditions. The conditions were optimized (Figure 7). Theoretical chemical formula: C110H156N26O29S. Observed ESI- MS m / z: 2402.6 -(M+1) +1 , 1201.9 -(M+2) +2 / 2(estimated Measured value: 2402.18)

[0095] 1.4.6: PD-L1 and PD-1 Binding Inhibition Assay: PD-L1 binding to PD-1 Competitive inhibition assays were performed in discussion with Cisbio (Woodard et al., 2014). , were optimized from a previously described fluorescence resonance energy transfer (FRET)-based assay. 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 The assay conditions were initially adjusted to the PD-1 and PD-L1 concentrations. PD-1-Ig at final concentrations of 10 nM, 20 nM, and 40 nM was used for with PD-L1-His-tag at final concentrations ranging from 0.65 to 320 nM (each concentration in triplicate). for 15 minutes, followed by anti-human IgG-Eu 3+ Cryptate (IgG-E u, final concentration 2 nM) and anti-6HIS-XL665 monoclonal antibody (anti-6HIS-X 10 μL of FRET buffer containing L665 (final concentration 40 nM) was added. After 1 hour of incubation, 1 μL of NaF assay buffer was added (final concentration, 40 0 mM), and the plate was loaded onto a Perkin Elmer Victor3 1420 multi-label using a counter (Perkin Elmer, Waltham, MA). I read it.

[0096] For competitive inhibition assays, inhibitors (WL12, WL12D and WL12-Cu 2 + , range: 1 pM to 1 mM) with PD-L1-His-tag (final 80 nM) in 10 μL Assay buffer for 15 min, followed by PD-1-Ig (final concentration 5 μL of assay buffer containing 20 nM of ATP was added and incubated for 15 minutes. Then, IgG-Eu (final concentration 2 nM) and anti-6HIS-XL665 (final concentration 40 nM) were added. 5 μL of assay buffer containing 1 μM was added. After incubation at room temperature for 1 hour, 1 μL of 1 L of NaF was added (final concentration 400 mM), and the plate was incubated in a Perkin Elmer V The data were read on a ictor3 1420 multilabel counter. By fitting the response curve and the Cheng-Prusoff equation, IC50 and The KD and Ki values were calculated, and a KD of 70 nM was derived 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 Cu By evaporating Cl2 to a small volume and titrating with 0.1 M sodium acetate solution 64 C For radiolabeling, approximately 1% HCl in 100 μL of sodium acetate was added. 0 μg of WL12D peptide conjugate (4.27 nmol) was added to approximately 185 MBq ( (approximately 5 mCi) 64 Mix with Cu(OAc)2 and incubate at 65°C for 30 minutes. The resulting radioactive tracer was analyzed by C-18 (Luna, 5 μm, 10 × 250 mm; Ph enomenex) semi-preparative column, equipped with a radioactive single-channel radiation detector. arian ProStar system (Model 105S; Bioscan, Poway, California) and a Varian ProStar UV absorbance set at 280 nm The product was purified using a temperature detector. % TFA) and reached 90% MeOH over 70 min at a flow rate of 5 mL / min. The elution was applied. 64 Cu]WL12 was collected at ~56.2 min (unlabeled peptide Retention time: 53.6 min), evaporated, diluted with saline containing 5% DMSO, and 2 drops Tween 20 was used for in vitro and in vivo evaluation. 64 C u]WL12 had a specific release of 1.9±0.11 mCi / μg with a yield of 52.09±6.3%. Obtained through radioactivity.

[0098] 1.4.7. Cell Line: Chinese hamster ovary cell line CHO-K1 (hereafter referred to as CHO) and triple-negative breast cancer (TNBC) cell line MDAMB231 were administered to Purchased from the American Type Culture Collection (ATCC, Manassas, VA). , passaged for less than 3 months, and then started a new culture from a vial of frozen cells. The 149 cell line was provided by Dr. Stephen P. Ethier of the Medical University of South Carolina. and verified by STR profiling at the Johns Hopkins Genetic Resource Facility. SUM149 cells were cultured in 5% FBS, 1% P / S, and 5 μg / mL insulin. and maintained in Ham's F-12 medium containing 0.5 μg / mL hydrocortisone. All cell lines were cultured in an ATC incubator at 37°C in an atmosphere containing 5% CO2. The cells were cultured in the recommended medium. They stably expressed human PD-L1 (hPD-L1). A CHO cell line that supports this has been generated in our laboratory (Chatterjee et al., 2016). in F-12K medium containing 10% FBS, 1% P / S, and 2% mg / mL G418. was maintained.

[0099] 1.4.8. Flow Cytometry: Cells in suspension are collected by centrifugation and adherent cells are collected. Cells were dissociated in enzyme-free PBS-based cell dissociation buffer (Thermo Fisher Scientific) Cells were detached using a centrifuge (Centific, Waltham, MA). Add 1x PBS containing 2 mM EDTA and 0.5% FBS to flow cytometry buffer (1x PBS containing 2 mM EDTA and 0.5% FBS). The cells were then washed twice with PBS (BS). The cells were then stained with phycoerythrin-conjugated anti-human PD-L 1 antibody (BD-MIH-PE, clone number MIH1, catalog number 557924, Bec ton Dickinson, Franklin Lakes, New Jersey) Flow cytometer stained according to the manufacturer's protocol and analyzed on a FACSCalibur The samples were analyzed using a Becton Dickinson meter. The event was recorded.

[0100] 1.4.9. In vitro binding: hPD-L1, CHO, MDAMB231 and [ 64 In vitro binding of [Cu]WL12 was measured using 1 μCi of radiation. 1x10 Sex Tracer 6 Determined by incubating with cells for 1 hour at 37°C. After incubation, the cells were counted in an automatic gamma counter (1282 Compugmam a CS, Pharmacia / LKB Nuclear, Gaithersburg Maryland Cells were washed three times with cold PBS before counting. 64 Cu]WL12P To demonstrate D-L1 specific binding, 1 μM of WL12 peptide or humanized anti-PD-L1 PD-L1 blocking was performed with the antibody atezolizumab. Mean fluorescence intensity values were measured using incubation All cellular uptake studies were performed in a controlled manner. Each cell line was performed in triplicate and repeated three times.

[0101] 1.4.10. Animal Models: Animal experiments were conducted by JHU Animal Care and The study was conducted in accordance with a protocol approved by the American College of Cardiology and Clinical Trials Use Committee (ACUC). Six to eight-week-old female non-obese diabetic severe combined immunodeficiency gamma (NSG) mice were cultured at JHU. Obtained from the Immune Compromised American Core. 10 x 10 cm on both sides of the upper abdomen 6 CHO-PDL1 and CHO cells were subcutaneously implanted. Tumors between 200 and 300 mm 3 When the volume reaches 100 μm, the mouse is imaged or in vivo It was used for intracellular distribution experiments.

[0102] 1.4.11. PET-CT Imaging of Mouse Xenografts: Mice were injected with 200 μL 150 μCi of [ 64 Cu]WL12 was injected intravenously (n=3) and Mice were anesthetized under 3% isoflurane before being placed in the canner. During imaging, mice were anesthetized under 1% isoflurane. PET images were taken at 2 bed positions for 10 min / bed. on the ARGUS small animal PET / CT scanner (Sedecal, Madrid, Spain). For anatomical coregistration, a CT scan was performed at the end of each PET scan. The PET data were analyzed using a two-dimensional regular subset-expectation maximization (EMM) algorithm. Reconstruction was performed using an algorithm (2D-OSEM) and the dead time and radioactive decay were analyzed. The %ID per cc values were calculated based on a calibration factor obtained from known radioactivity. Final data visualization and image generation was performed using Amira® (FEI This was achieved using a 3D printer (Hillsboro, Oregon).

[0103] 1.4.12. Ex vivo biodistribution: high and low PD-L1 expression ( n=5) and CHO tumor-bearing mice were treated with 40 μCi of [ 64 C u]WL12 was injected intravenously. 64 Cu] 1 and 2 hours after WL12 injection. , blood, tumor, and selected tissues were collected, weighed, and analyzed using an automated gamma counter (Per Elmer-2480 Automatic Gamma Counter - Wizard2 3´´ Walla For the blocking test, mice were given 2 mg / kg (50 μg) of untreated The peptide was co-injected with the radiotracer. Percentage of injected dose per gram of tissue The percent ID / g values were calculated using triplicate measurements, signal decay corrected and external [ 64 Cu] standard Biodistribution data shown are mean ± standard deviation of the mean. Errors are mean average mean squared error (SEM).

[0104] 1.4.13. Data Analysis: Prism 6 software (GraphPad software) Statistical analysis was performed using an unpaired two-tailed t-test using the National Institute of Health Sciences (NIHSS, La Jolla, CA). A P value of <0.05 was considered significant, and comparators were selected as those with low PD-L1 expression. Flow cytometry data were analyzed using FlowJo software. Analysis was performed using an IC analyzer (Tree Star, Ashland, Oregon). 50 Oh The K 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 been shown to produce durable responses in a variety of malignancies. However, immune checkpoint targeting therapy has improved patient survival rates. nearly 70% of patients treated with the method do not respond to monotherapy ( Lipson et al., 2015 ; Topalian et al., 2015). Identifying determinants of response to precision immunotherapy There is an unmet need. Combination checkpoint therapy improves survival, but In many cases, increased knowledge of combination strategies is beneficial at the expense of increased immune-related adverse events (irAEs). suggest that addition is necessary to reduce toxicity (Marrone et al., 2016 ) New biomarkers for immune checkpoint therapy and their breadth and durability Intensive research is needed to identify combinations of these drugs that enhance efficacy and reduce irAEs. Therefore, one aspect of the presently disclosed subject matter is the development and Develop strategies for using PD-L1-based PET imaging in the detection and evaluation of Plasma- or tissue-based (biopsy) therapy is invasive and impractical in advanced-stage patients. Unlike current strategies that rely on biomarkers, the present invention aims to develop a PD-L1-targeted therapeutic agent (antibody). Dose-occupancy relationships of drugs (antibodies, peptides, small molecules) in relevant in vivo tumor models and establish using PD-L1 PET imaging.

[0106] Advances enabled by PET-based quantification of PD-L1 dynamics: NSC PD-L1 Targeted Therapeutic AtzMab and Its Mouse Kidney Cancer Cell Line in LC, TNBC, and Colon Tumors It has recently been discovered that the accumulation of melamine (PRO) is not entirely dependent on PD-L1 expression. However, H2444 NSCLC xenografts with high PD-L1 expression were detected by IHC and Breast cancer xenografts with low PD-L1 expression as detected by flow cytometry Because significantly less radiolabeled AtzMab accumulated relative to that seen in the explants (Chatterjee et al., 2016). Similarly, in a syngeneic mouse tumor model Systemically injected radiolabeled PRO was primarily associated with tumor vasculature and distributed to tumor parenchyma. showed little or no diffusion into tissues (Deng et al., 2016). This finding may be due to pathophysiological features including elevated interstitial pressure within the tumor (Baxter et al., 2013). er et al., 1989; Baxter et al., 1990), which prevents the accumulation of therapeutic drugs within tumors, This is an important factor in treatment resistance (Goel et al., 2011). This will allow PD-L1-targeted therapeutic agents to act on tumor cells and tumor immune infiltrates to gain access to tumor cells. This could potentially hinder the 18 F]WL12 and other peptides Due to their very small molecular size, radiolabeled peptides, such as ribonucleotides, are often used as antibodies. It can penetrate tumor tissue and reach target cells more effectively and efficiently than conventional drugs. By using the analysis and correction 18 F]WL12 measurement or similar radioactivity Measurements performed using labeled peptides reveal the desired occupancy in tumor tissue in target tumor cells. This may help identify / optimize the therapeutic mAb dose required to achieve

[0107] Therefore, PD-L1-directed PET has been applied to the development of PD-L1-targeted drugs. In order to assess the potential value, innovative strategies [ 64 Cu]WL12, Therapeutic PD-L1 antibody atezoline in relation to dose versus mAb localization in tumors as seen by PET We evaluated and compared the tumor PD-L1 engagement characteristics of tafamidis (AtzMab) The preclinical findings disclosed herein may have clinically actionable implications. used similar PD-L1 PET-based imaging measurements to guide treatment dose intensification. , which may improve the therapeutic effect (Yang et al., 2013; Oude M Moreover, such PD-L1 PET measurements can be used to detect tumor-specific PD-L1 expression in tumors. By enabling quantification of their potential target engagement in This may guide the future development of novel PD-L1-targeted therapeutics.

[0108] 2.1.2. Innovations in the use of PD-L1 PET in drug development and evaluation: The innovative PD-L1 peptide-based PET imaging strategy presented here is a promising candidate for current and future Target engagement efficacy of anti-PD-L1 therapeutics in the tumors where they are most relevant Enables assessment of dynamic PD-L1 density / turnover (i.e., occupancy and residence time). The extent of PD-L1-expressing tumor burden, which influences serum mAb concentrations, is important for complete tumor perfusion. This, together with the overall efficacy and resulting intratumoral mAb accumulation, significantly impacts therapeutic efficacy. Radiolabeled antibodies were used to define the required mAb dose level and to calculate target surface molecule occupancy. Although it has been previously used to detect and treat cerebrospinal fluids (Deng et al., 2016), there are important limitations to that approach. The approach of the present disclosure can only predict PD-L1 occupancy at the tumor site of action. This effectively addresses this issue and quantifies PD-L1 occupancy at the tumor site. The relationship between effective mAb dose and cumulative dose achieved versus key tumor physiological parameters was investigated. In addition to considering the contribution of our novel PET tracer base Measurement of PD-L1 activity may explain why some patients with PD-L1-positive tumors do not respond to CIT. Improving current understanding of the drug and increasing doses to achieve desired tumor occupancy levels I hope it will provide some strategic guidance.

[0109] (2.1.3. Usefulness of PD-L1-PET in the development and evaluation of PD-L1-targeted therapeutics Rate the sex :) (2.1.3.1 Rationale) Therapeutic antibodies targeting PD-L1 and PD-1 are being used in patients with PD-L1-positive tumors. At currently used doses, it has shown excellent efficacy in a small proportion of patients. Responder and non-responder populations showed approximately 65% PD-L1 occupancy in PBMCs. However, the relationship between PD-L1 occupancy in PBMCs and tumors is dynamic. However, this is not fully understood (Brahmer et al., 2012). The study found that in some tumors, PD-L1 antibodies were restricted to the tumor vasculature. Preliminary results using radiolabeled AtzMab demonstrated that NSC summarized these findings in LC xenografts ( Chatterjee et al., 2016 ). Taken together, these findings highlight the role of PD-L1 occupancy in tumors and its dose-dependent role. Improving our understanding of the residence time of anti-PD-L1 antibodies in tumors will improve This suggests that informed PD-L1-directed therapy is necessary. Without wishing to be bound by any of the above, PD-L1 PET is a promising treatment option for patients with PD-L1 Such PK measurements of peptides and small molecules are evaluated with respect to target binding and residence time. PET-based administration is also considered to be a valuable tool for PD. PD-L1 PET can be used to quantify tumor PD-L1 expression and immune cell infiltration. We also investigated changes in the immune profile within tumors that can be correlated with treatment-induced changes in the immune system. It is thought that this will lead to

[0110] 2.1.3.2 Representative Data: Emission of Available Anti-PD-L1 Antibodies and PD-1 Derivatives The labeled version has been used to noninvasively detect PD-L1 expression (Cha tterjee et al., 2016;Deng et al., 2016;Hettich et al., 2016;J osefsson et al., 2016; Lesniak et al., 2016; Heskamp et al., 20 15; Maute et al., 2015). To this end, the therapeutic antibody AtzMab is The specificity of its PD-L1 detection was confirmed by selecting for cross-reactivity in immunocompromised and immunosuppressed mice. Human TNBC and NSCLC xenografts in mice and the 4T1 syngeneic breast tumor model In sections, this was demonstrated by PET, SPECT and optical imaging (Chatt erjee et al., 2016; Lesniak et al., 2016) (Figure 20A, Figure 20B, Figure 20 C, and Figure 20D). AtzMab showed high affinity to both human and mouse PD-L1. The dissociation constants (Kd) are 0.43 nM and 0.13 nM, respectively. Irving et al., 2012; Powles et al., 2014) AtzMab is a novel treatment for progressive or Metastatic bladder cancer (Powles et al., 2014) and melanoma (Hamid et al., 2013) NSCLC, (Spigel et al., 2013) RCC, (Cho et al., 2013) TNBC, and It is under clinical evaluation for the treatment of cancer and several other cancers.

[0111] Accumulation of radiolabeled AtzMab in tumors was observed in both cancer types (NSCLC and TN) BC) was found to be PD-L1 specific (Chatterjee et al., 2014). 016; Lesniak et al., 2016). 111I n]AtzMab accumulation in tumors , and was found not to be completely dependent on PD-L1 expression, which was due to factors such as interstitial fluid pressure, tumor convection, suggest that spatial variation in blood flow and extravasation are some of the contributing factors. This is a problem often observed with antibodies (Baxter et al., 1989). 31 TNBC xenografts than subcutaneous and orthotopic H2444 NSCLC tumors Tissue accumulation (percentage of injected dose per gram; %ID / g) was measured by flow cytometry. showed high PD-L1 expression by both cytometry and IHC analysis (Chatterje (e et al., 2016). Our novel peptide-based PD-L1 PET tracer By taking advantage of specificity and flexibility, we have demonstrated that PD- We analyzed the kinetics of AtzMab accumulation in L1-expressing tumors and investigated the various mechanisms that influence antibody distribution within the tumor. A completely different approach accounting for multiple factors applies to various PD-L1-targeting antibodies It is possible.

[0112] 2.1.3.3 Accumulation of PD-L1 therapeutic antibodies in tumors by PD-L1 PET: PD- While assessing the specificity of WL12 for L1, WL12 binds to the same binding site on PD-L1. It was found to compete with AtzMab for the PD-L1-directed PE Using T, novel and previously unrecognized methods to evaluate AtzMab therapy at tumor sites where needed This provides an unexpected tool for understanding the distribution of PD-L1 antibodies in tumors. Improvements may have implications for clinical antibody administration and therapeutic monitoring. Therefore, we evaluated the binding of AtzMab to PD-L1 in tumors. 64 Cu]WL12-P ET's capabilities were tested. 64 Cu]WL12-PET and biodistribution studies As quantified, the accumulation of radioactivity in hPD-L1 tumors was significantly higher with AtzMab (20 mg / kg) (Figures 21A, 21B, and 2 1C). The radioactivity uptake in tissues other than the kidney was reduced, but no significant difference was observed. 64 Cu]WL12 binding was shown to be specific to human PD-L1 (Lesniak (2016). In vitro binding studies showed that unlabeled WL12 binds to C Concentration-dependent inhibition of y5-conjugated AtzMab binding to PD-L1 was observed, with IC 50 37.8 nM, and both ligands compete for PD-L1 binding (Figure 21D ), AtzMab is [ 64 Cu] is more potent than WL12 in inhibiting WL12 binding. This allows detection of unoccupied PD-L1 levels in tumors upon administration of AtzMab. In summary, these results confirm that the binding of WL12 and AtzMab is Demonstrating overlapping sites, AtzMab target enzymes in PD-L1-expressing tumors to assess engagement and dwell time (target engagement efficacy) 64 This demonstrates the potential utility of [Cu]WL12-PET. The applicability of this approach is The results also extended to cancer cell lines where IL-1 expression was naturally elevated. To detect AtzMab accumulation in high PD-L1-expressing MDAMB231 xenografts 64 The ability of [Cu]WL12 to inhibit the 20mg / Kg AtzMab dose was observed (Figure 22). A significant decrease in PD-L1 PET imaging agent uptake was also observed in mice receiving the treatment.

[0113] It has similar applications to other PD-L1-targeting therapeutic antibodies such as Avelumab (AvMab). AvMab is a promising treatment for NSCLC (NCT02395172), advanced RCC, and Human IgG1 is currently undergoing multiple Phase III clinical trials in several cancers, including gastric cancer. Analysis of the crystal structure of PD-L1 complexed with AvMab revealed that A vMab binds to some of the same amino acids on PD-L1 (R113, D 61, and E58) (Liu et al., 2016), which suggests that A In vivo targeting by vMAbs and possibly other PD-L1-directed therapeutic mAbs Potentially advantageous uses of WL12-based tracers for assessing bat engagement These studies demonstrate the utility of PD-L1 PET in patients undergoing ongoing PD-L1 mAb therapy. This paper examines the possibility of evaluating laws in terms of their target engagement capabilities.

[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 shown to Nearly one-quarter of clinical trials involving checkpoint inhibitors are in place. levels, occupancy by these PD-L1 therapeutics, and ensuring optimal immune responses The relationship between the dose and the extent and duration of target engagement within the tumor for The occupancy of PD-L1 in tumors is a function of the dynamic changes in PD-L1 expression, if any. and by tumor-intrinsic and tumor-extrinsic parameters that alter plasma and tumor antibody concentrations. However, such significant variations may be due to peripheral pharmacokinetic and pharmacodynamic assessments. To address the gap in the relationship between PD-L1 expression and PD-L1 signaling, we aim to quantify dynamic changes in PD-L1 expression. Radiolabeled PD-L1 binding peptides were investigated. Structural analysis was performed on the peptides and therapeutic monoclonal antibodies. showed overlap in the interactions of mAbs with PD-L1 and therapeutics in tumors The occupancy of the mAb could be measured using positron emission tomography (PET). PET imaging and biodistribution studies in multiple xenograft models have shown variable We demonstrate that PD-L1 expression and its saturation by PD-L1 therapeutic antibodies can be quantified. Furthermore, we measured PD-L1 occupancy in tumors using three different antibodies and We quantified the effects of dose and time on PD-L1 occupancy in peptide-based P2000 cells. D-L1 PET can be used to refine dose and treatment regimens with the goal of reducing immune-related adverse events. It is a promising tool for optimization.

[0115] More specifically, the subject matter of the present disclosure relates to quantitative positron emission tomography (PET) imaging. Using PD-L1 signaling, we investigated the PD-L1 expression levels and PD-L1 expression in tumors in vivo. Addressing the need to characterize mAb target engagement, which is essential in tumors Repeated measurements of target expression in the lab (Wilman et al., 2008) and are useful for drug development and evaluation. However, it is rarely used for receptor occupancy studies in oncology (Rathkopf et al. , 2013), particularly the pharmacokinetic and pharmacodynamic evaluation of PD-L1 or PD-1 mAbs. The efficacy of this approach has not been achieved (Peterson et al., 2008; Linden et al., 20 06).

[0116] It binds to human PD-L1 with high affinity and specificity, and is one of the first therapeutic targets for radiotracer administration. Generates high-contrast images within 20 minutes 64Small peptides radiolabeled with Cu , [ 64 Cu]WL12 was recently developed (Chatterjee et al., 2017). The example shows a method for detecting PD-L1. 64 Cu]WL12-PET was described and used to evaluate lung cancer and Quantifying dynamic changes in PD-L1 expression in experimental breast cancer models. Three different FD A-approved mAbs, atezolizumab, avelumab, and durvalumab (DurMab) Evaluate PD-L1 engagement in patients with PD-L1-associated leukemia 64 The capabilities of Cu]WL12 PET were evaluated. Furthermore, the relationship between the extent and duration of PD-L1 engagement in tumors and PD-L1 activity was examined. The L1 mAb dose association was assessed noninvasively.

[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 (PDDL). PD-L1 acts as a means to evade tumor-infiltrating cytotoxic T cells. It is expressed by many tumors (Topalian et al., 2016) and binds to the PD-1 receptor. It causes immunosuppression through direct binding (Okazaki et al., 2007; Topalia (N et al., 2015). Multiple PD-L1-targeting monoclonal antibodies inhibiting PD-L1:PD-1 interactions. Clonal antibody therapeutics (mAb) are currently in clinical trials, and patients receiving these treatments Nearly 30% have a sustained response (Topalian et al., 2015; Lipson et al., 2 015). However, despite these successes, clinical challenges remain. Abnormalities such as delayed or mixed tumor regressions that limit clinicians' ability to proceed with point-of-care therapy There is an incomplete understanding of the biological mechanisms that contribute to these response patterns.

[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) and are limited Only a limited number of studies have reported these results, which were obtained using peripheral blood mononuclear cells (PBMCs). The PD-L1 antibody BMS-936559 is being studied at doses ranging from 0.1 to 10 mg / kg. Uniform target occupancy of 64–70% has been reported for this dose (Brahmer et al., 2014). PD-L1 mAb placement in the most relevant location, i.e., tumor, and and the degree and duration of target engagement to ensure an optimal immune response. Much remains unknown about the relevance of administration between

[0119] The most studied predictive biomarkers for therapeutic monitoring of PD-L1 / PD-1 targeted therapies The tumor marker was PD-L1 immunohistochemistry (IHC) (Gibney et al., 2016). However, the method requires biopsy specimens, which are of limited availability and are temporally dynamic. Positively assess the immune tumor microenvironment (TME) and intra- and inter-tumor heterogeneity of PD-L1 expression have significant limitations as they may not accurately reflect the aughlin et al., 2016). PD-L1 expression levels in primary and metastatic tumors Non-invasive assessment of tumor kinetics, pharmacokinetics, and disposition of PD-L1 therapeutic agents, as well as imaging targets There is an unmet need to do so within standard clinical workflow.

[0120] (3.3 results) 3.3.1: Structural analysis and characterization of PD-L1 interactions with WL12 and PD-L1 mAbs WL12 inhibits PD-L1:PD-1 interaction with high affinity (IC5 It is a 14-amino acid peptide that inhibits ATP at 0:20 nM (Chatterjee et al., 20 17) Initial molecular modeling analysis has identified PD-L1:W as a key molecular interaction. L12 and PD-L1: PD-1, four amino acids of PD-L1 (Y56, E58, D 61 and A113) suggested an overlap in the interaction surfaces (Chatterjee et al., 2017). P complexed with the therapeutic antibody atezolizumab (AtzMab) The burial surface of D-L1 (2,106 Å 2 ) is larger than that of PD-1 (1,970 Å 2 ) (Lee et al., 2017). While not wishing to be bound by any one particular theory, The WL12 interaction surface on PD-L1 also overlaps with that of clinically available therapeutic mAbs This is thought to be because they are similarly designed to inhibit PD-L1:PD-1 interactions. To test this, we calculated the predicted binding conformation of WL12. The results were compared with those of PD-L1 mAbs. All mAbs, as well as PD-1 and WL12, The overlapping AA contacts of PD-L1 residues Y56, E58, A113, M115 and Y1 23 reveals a common binding domain. Visualization of the PD-L1 molecular surface (Figure 33 As revealed in Figure 34A, the overlapping region (cyan) is a deep pocket. and acts as an anchor point for all interaction points. zMab (red) binds PD-1 (purple), WL12 (green), and avelumab (AveMab, range), and durvalumab (DurMab, blue). PD with loops from antibodies that make molecular contacts with residues on all sides of a common binding core -L1 interacts more with the surface.

[0121] To support the aforementioned structural analysis, Cy5-labeled AtzMab, AveMab, and Du rMAb was conjugated to commercially available Cy5 fluorescent N-hydroxysuccinimide esters of antibodies. CHO cells (Cho- hPD-L1) and MDAMB231 breast cancer cells that naturally express PD-L1 (Chat A competitive inhibition assay with WL12 was performed (Terjee et al., 2016). WL12 dose-dependent inhibition of Cy5-PD-L1 mAb binding to PD-L1 at inhibitory concentrations of 100 μM The adverse effects were observed in HCC827 and H226 non-small cell lung cancer (NSCLC) ) cells, each of which naturally expresses PD-L1, in the presence of 5 nM WL12. Incubation with fluorescent versions of AtzMab, AveMab and DurMab. Flow cytometry showed a significant decrease in binding fluorescence (P<0.001), indicating that antibody-PD-L The ability of WL12 to disrupt CX1 interactions was further demonstrated (Figures 34C and 34D). No change in binding fluorescence is observed when using the CR4-specific antibody MDX1338 This provided further confirmation of the specificity of the WL12:PD-L1 interaction. (HCC827, H226, MDAMB231, and hPD-L1) and PD-L1 Negative (Sum149 and CHO) cells were treated with WL12 analogs radiolabeled with 64Cu ( [ 64The WL12 analogs were incubated with hPD-L1 / High affinity (IC50<2 in vitro and in vivo in CHO cells) It has previously been demonstrated to bind PD-L1 with high potency (0 nM) and selectivity, but with variable expression. It has not been tested in human cancer cell lines (Chatterjee et al., 2017). Compared with PD-L1-negative cells, [ 64 Cu]WL12 high A significant expression-dependent uptake was observed (P<0.0001). As a further check on efficacy, the 60 nM mAbs were compared to PBS-treated controls. When treated, the [ 64 Cu]WL12 uptake significance Significant blockade (P<0.0001) was observed (Fig. 33C). 64 Cu]WL12 was used to detect free PD-L1 levels in tumors and to detect PD-L1 levels by PD-L1 mAb. 1 This shows that engagement can be monitored.

[0122] 3.3.2 Quantification of tumor PD-L1 engagement by AtzMab. Noninvasive in vivo evaluation of PD-L1 engagement by therapeutic mAbs To investigate the efficacy of NSCLC in NSCLC xenograft models, we tested these models. Approximately 50% were PD-L1 positive, and PD-L1 IHC was negative for patients receiving immune checkpoint therapy. It was selected because it is used as a predictive biomarker for NSCLC patients with Mansfield et al., 2016). H showing low and intermediate PD-L1 expression, respectively. NOD scid gamma mice bearing 226 and HCC827 cell-derived xenografts ( Figure 36A) was compared with a single dose of intravenously administered AtzMab (20 mg / kg, 24 h) They were processed in [ 64 PET images acquired 2 hours after [Cu]WL12 injection show H in HCC 827 tumors compared with 226 [ 64 Cu] WL12 There was a clear reduction in the accumulation of radioactivity in the tumors of AtzMab-treated mice, The results show a reduction in the levels of available PD-L1 sites compared to treatment controls (Figure 35A and and Figure 35B). PET imaging results were supported by ex vivo measurements of biodistribution. This was further confirmed (Figures 35D and 35E, 36B and 36C), which is consistent with the results of saline vs. The [per gram of injected dose] in AtzMab-treated mice compared to controls 64 Cu]W L12 showed a significant decrease in percent (%ID / g): H226-bearing mice 34% in HCC827 xenografts (P<0.0001) and 47% in HCC827 xenografts (P<0. 001). PD-L1 expression levels were confirmed by PD-L1 IHC in xenografts. (Figures 35C and 35F). The results are 64 Cu]12 was used to demonstrate that in vivo targeting of PD-L1 in tumors can be quantified .

[0123] Efficacy of a single dose of AtzMab to target different PD-L1 levels in tumors To evaluate efficacy, we used C4700 cells with PD-L1 expression 4-10 times higher than NSCLC cells. PET and biodistribution studies were performed in tumors derived from the HO-hPDL1 cell line (Figure 36) CHO-hPDL1 / C cells treated with AtzMab (20 mg / kg, 24 hours) HO tumor-bearing mice showed a significantly higher [ 64 Cu]W Biodistribution studies showed a significant decrease in L12 uptake after AtzMab treatment (Figure 35G). In CHO-hPDL1 tumors compared with conventional tumors, 64 Cu] 77% of WL12 bonds showed a decrease in tumor PD with AtzMab (Fig. 35H, Fig. 36D) (P<0.0001). We demonstrated the measurement of PD-L1 targeting in PD-L1-negative CHO tumors with low levels of [ 64 Cu WL12 uptake was observed in hPD-L1 tumors treated with AtzMab. These observations were similar to those in hPD-L1 and CHO tumors. This was confirmed by the observation of strong and weak immunoreactivity, respectively (Fig. 35I). 64 Cu]WL12-PET can detect graded levels of PD-L1 expression in tumors A single 20 mg / kg dose of AtzMab significantly increased PD-L1 levels across a wide range of tumors. Demonstrate that it can be involved in

[0124] 3.3.3. Quantification of dynamic changes in PD-L1 expression. PD-L1 is expressed by various cytokines. Interferon contributes to the dynamic and spatiotemporal heterogeneity in PD-L1 expression, especially It is known to be upregulated in response to interferon gamma (IFNγ) (Taube et al., 2014). 015; Taube et al., 2012). Inducible PD-L1 expression in tumors in vivo We evaluated the robustness of [64Cu]WL12 quantification and its effect on AtzMab treatment. Blocking such upregulated PD-L1 [ 64 Cu]WL12-PET It was determined whether the serotonin levels could be increased (Figures 37A, 37B, 37C, 37D, 37E, and Figure 37F).

[0125] To do so, we investigated the effects of doxycycline-induced PD-L1 expression on A549 NSCL A549 expressed PD-L1 at baseline. The G12S lung adenocarcinoma cell line expresses low levels of Kras. PD-L1 in the virus pINDUCER20 vector (Meerbrey et al., 2011) Transduce cells, select with G418, and confirm PD-L1 induction by flow cytometry ( (Figure 37A) and was used in in vitro and in vivo studies. Binding of Cy5-PD-L1-mAb to phospho-treated A549-iPDL1 cells was compared with WL12 This was blocked by WL12, demonstrating its specificity (Fig. 37B). 64 Cu ] Incubation with WL12 compared doxycycline-treated vs. untreated cells and P D-L1 showed a 5.5-fold increase in radioactivity uptake in low-density A549 cells (P<0.05). .0001). Doxycycline-treated A549-iPDL1 cells 64 Cu]WL1 2 binding was significantly reduced in the presence of 60 nM AtzMab, AveMab, and DurMab. These in vitro studies showed a significant reduction in IL-1 expression (65%, P>0.0001) (Fig. 37C). , in A549-iPDL1 NSCLC tumors after 72 h of doxycycline treatment [ 64 Cu]WL12 accumulation was 65% higher than in A549 control tumors. This was confirmed by in vivo studies (P>0.0001). 64 Cu]WL12 uptake increase 64 Cu]WL12-PET and biodistribution studies As quantified by ab-treated group, the tumors were reduced by >75% (Fig. 37D and Fig. 37E). IHC analysis of the tumors revealed that A5 49-iPDL1 showed a strong PD-L1 signal, but not A549 tumors. The results confirmed the sizing and biodistribution results (Figure 37F). , to detect dynamic changes in PD-L1 expression levels [ 64 Cu]WL12 possibility, and A We have demonstrated its blockade by tzMab. PET is expected to play an important role in quantifying dynamic changes in PD-L1 expression. , providing new ways to inform treatment decisions.

[0126] 3.3.4. Quantification of tumor PD-L1 engagement with different antibodies. Radiolabeled Anti-PD-L1 antibodies have been developed and are effective in human tumor xenografts and syngeneic mouse tumor models. Their potential to noninvasively assess PD-L1 expression has been demonstrated (Chatter Jee et al., 2016;Heskamp et al., 2015;Maute et al., 2015;Deng et al., 2016; Hettich et al., 2016; Josefsson et al., 2016). Radiolabeled antibody conjugates such as these are currently used to detect PD-L1 (NCT02453 984) and imaging other tumor-specific proteins (Gebhart et al., 2016) Although they are used clinically to determine antibody kinetics, their routine clinical application is limited. To enhance contrast and lesion detection (Pandit-Taskar et al., 2015; Oosting et al., 2016), faster clearance times (hours vs. days) ) is required (Wu, 2014). A further limitation is that the radioactive Observations made with labeled antibodies are highly specific to the antibody under investigation and can vary in valency, shape, size, etc. It is determined by antibody properties such as length, isoelectric point, and dosage, each of which affects its pharmacokinetics. These unique biophysical characteristics of mAbs also affect plasma half-life, tissue exposure, and ultimately impact efficacy. (i) PD-L1 antibody target engagement (ii) takes into account the characteristics of mAbs; and (iii) is applicable to all antibodies. A new approach is needed.

[0127] For each of the three FDA-approved antibodies, AtzMab, AveMab, and DurMab Quantifying PD-L1 engagement in tumors non-invasively using 64 Cu]W The ability of L12-PET was evaluated. NSG mice bearing MDAMB231 tumors were cultured in Atz Mab, AveMab, or DurMab, and then 24 hours later of[ 64 Cu]WL12-PET (Figures 39A, 39B, 39C, and Figure 39D). In all treated mice, tumors showed significantly higher IL-1 expression compared to saline controls. Low signal, low levels of free PD-L1 from tumor PD-L1 engagement and Radiotracer blockade by mAbs was confirmed. Ex vivo quantification of tumors confirmed these This confirmed the findings of [ ] and showed that at 120 min post-injection, [ 64 Cu]WL demonstrated approximately 60% less uptake of 12 compared to the saline control (Figure 39E). IHC analysis of saline controls showed moderate to high PD-L1 intensity in tumors ( Figure 39F). The results show that tumor PD-L1 engagement by PD-L1 therapeutic mAbs Despite the different biophysical properties, plasma and tissue kinetics of each antibody, [ 64 Cu]WL It has been demonstrated that quantification is possible using 12-PET.

[0128] 3.3.5. Effect of dose on PD-L1 occupancy in tumors. Antibody kinetics in tumors is governed by both intrinsic and extrinsic parameters of the tumor (Ago In recent years, factors other than PD-L1 expression itself have been implicated in NSCLC, TNBC, and PD-L1-targeting therapeutic agent AtzMab and its mouse chimera (PRO3) in human and colon tumors It was found that the accumulation of 04397) can be reduced (Chatterjee (2016). Furthermore, at doses below 1 mg / kg, systemically injected radiolabeled anti-P The D-L1 antibody PRO304397 was primarily associated with tumor vasculature and inhibited PD-L1-expressing syngeneic mouse tumors. showed minimal diffusion into the tumor parenchyma in tumor models (Deng et al., 2016). These findings may be due to factors such as increased interstitial pressure within the tumor (Baxter et al., 2013). et al., 1989; Baxter et al., 1990), which may contribute to resistance in tumors. mAb accumulation (Goel et al., 2011). Such effects also This may hinder the access of larger PD-L1-directed agents to tumor cells and immune infiltrates. Measurement of D-L1 and PD-1 therapeutic occupancy has not been reported in tumors and has not been performed using PBMCs. The evaluation is limited to the use of

[0129] To evaluate the effect of dose on tumor PD-L1 occupancy in tumors, MDAMB2 31 tumor-bearing mice were treated with increasing doses of AtzMab, ranging from 0.009 to 24 mg / kg body weight. After 24 hours, 64 Imaging was performed 2 hours after injection of [Cu]WL12 and Biodistribution studies were performed. PET images of mice receiving 0.06 mg / kg were compared with untreated Compared to the control [ 64 Cu]WL12 uptake in tumors, and no difference was observed in the The PD-L1 occupancy rate was low (Figure 41A). At each dose, there was a proportional decrease in signal intensity in the tumor, with a 3.2 mg / kg dose In this study, nearly 100% target engagement with the tumor was demonstrated by the antibody.

[0130] The radioactivity accumulated in the tumor (%ID / g) was then used to calculate the inhibitory sigmoid E max The model was fitted. The %ID / g data were compared with the AtzMab used in our experiments. The dose of the peptide radiotracer [ 64 Cu]WL12 was used to detect tumors The relationship between the reduction in free PD-L1 ligand in the PD-L1-positive patients and the reduction in free PD-L1 ligand in the PD-L1-positive patients was adequately fitted and explained (Figure 41 B and Figure 41C). 50% of maximum PD-L1 engagement in tumors (ID 50 ) or maximum fractional reduction of free PD-L1 ligand from baseline (I max ) Cause The dose of AtzMab was estimated to be 0.43 mg / kg (Table 2). ma x ID involved in 90% and 96% of 90 and ID 96 are 0.87 mg / These dose levels corresponded to 1.19 mg / kg and 1.19 mg / kg, respectively. This is comparable to the 1 mg / kg dose reported by Denget et al. (Deng et al. , 2016). Anti-PD-L1 antibodies and chimeric anti-PD-L1 antibody PRO304397 (21 ) for the same average 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 are tentatively estimated at 59 nM (8.6 mcg / m L), 120nM (17.4mcg / mL) and 164nM (23.8mcg / mL) These results were based on measurements performed in the tumor for dose selection and optimization. This shows the possibility of using a constant value.

[0131] The interaction of antibodies with their targets involves the stabilization or internalization and activation of PD-L1 by antibody binding. The study is expected to be a small step forward in that it may affect the natural dynamics of PD-L1, including the development of therapeutic antibodies. Unlike the interactions of antibodies, these interactions can have a significant impact on the tumor and serum kinetics of antibodies (Table In early pharmacokinetic studies of AtzMab, the doses were 0.6-1 mg / kg. Nonlinear PK below 1 mg / kg and linear PK above 1 mg / kg have been reported, resulting in ATA. A trend toward decreased serum antibody concentrations was observed in patients with the disease (Stroh et al., 2017). However, such tumor-intrinsic factors affect the PK and occupancy of PD-L1 antibodies in tumors. The influence of sex and extrinsic parameters is unknown.

[0132] [Table 2]

[0133] Detecting temporal changes in antibody dynamics in tumors 64 Cu]WL12-PET capability To investigate this, NSG mice bearing MDAMB231 tumors were treated with nonlinear and linear Injections of AtzMab at doses of 0.6, 10, or 20 mg / kg produced phenotypic kinetics. PET imaging and biodistribution studies were performed at 24 and 120 hours. was also reflected in tumor uptake values in all three dose groups compared to untreated controls. [ 64 There was a significant decrease in [Cu]WL12 uptake (Figures 41D and 41E). At 0 hours, the 0.6 mg / kg dose group showed a significant improvement compared with 24 hours. 64 Cu]WL12 uptake In contrast, in the 10 or 20 mg / kg treatment groups, 64 Cu] There was no significant time difference in WL12 uptake. At 120 hours, [ 64 Cu]WL12 uptake was similar in the 0.06 mg / kg treated and saline control groups; This suggests drug elimination from the tumor and reflects the nonlinear PK of AtzMab at low doses. Results show a dose- and time-dependent response to PD-L1 engagement in a mouse model. Both existential changes 64 Cu]WL12-PET.

[0134] 3.3.6. Discussion Immune checkpoint therapeutics are being tested in hundreds of clinical trials, of which approximately 25 % target PD-L1. Only 30% of patients receiving PD-L1 therapy To respond to treatment, the molecular and cellular basis of response and resistance to these therapies is being investigated using transcriptional, genetic, and epigenetic studies. The relationship of dose to drug accumulation and target saturation in relation to efficacy is unknown. Therefore, the large size of antibody therapeutics limits tumor penetration and limits specific pharmacodynamic evaluation at the site of action. The challenge is to consider both tumor-intrinsic and tumor-extrinsic parameters and to provides real-time PD-L1 saturation / occupancy data and is widely applicable and effective This lack of knowledge has impacted dose selection, dose optimization, treatment development, and toxicity. In our current study, radiolabeled PD The PD-L1 binding peptide can noninvasively detect variable and dynamic PD-L1 expression levels. Tumor intrinsic parameters (PD-L1 expression, recycling, interstitial pressure) and extrinsic parameters To measure tumor occupancy, taking into account factors such as antibody isotype, kinetics, ATA, and catabolism can be used to inhibit PD-L1:PD-1 interactions in tumors, thus It has been shown to provide a universal means for monitoring therapeutic activity in the body.

[0135] IHC-based clinical trials have been developed to assess PD-L1 expression in tumors Although there have been studies (Herbst et al., 2014; Roach et al., 2016; Meng et al., 20 15), PD-L1 IHC takes into account only a small proportion (0.1%) of single lesions. PD-L1 expression in the tumor microenvironment is spatially and temporally heterogeneous and may contribute to immunotherapy responses. Such an approach has significant drawbacks, as the response is inherently delayed, complex, and abscopal. Additionally, tissue samples obtained by biopsy for testing are typically very are limited to other pathways that confer sensitivity or resistance to existing therapies (e.g., B to identify targetable oncogenic mutations in BRCA1, BRCA2, and PARP These valuable data may be required for molecular profiling (Nolan et al., 2017). Samples should undergo multiple PD-L1 assessments for reliable delineation of PD-L1 expression. These problems often make immune checkpoints impractical (Gibney et al., 2016). worsening in patients with metastatic disease, a population for which point therapy has 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 allows for rapid assessment of the TME. Development of PET radiotracers that enable quantification is required. 64 Cu]WL12 The disclosed study demonstrates that variable and dynamic changes in PD-L1 expression are important predictors of progression-free survival in patients with advanced PD-L1 disease after standard clinical trials. Quantifiable within the workflow and have important clinical implications for patient selection and monitoring treatment Demonstrate that it brings

[0136] PD-L1 therapeutic antibodies have become important drugs in cancer immunotherapy. For CNS diseases, in vitro binding affinity measurements and occupancy studies are useful for dose selection. It is routinely used for the selection of drugs and prediction of pharmacological response (Lee et al., 2006). However, large molecules such as antibodies can be identified based on in vitro binding affinity. presents unique challenges in predicting receptor occupancy in vivo (Agor am, 2009). Antibody concentrations in tumors depend on antigen density and turnover, tumor burden, and tumor size. Several tumor-specific parameters, such as tumor perfusion, limit the penetration of mAbs into the tumor. Tumor and plasma concentrations of mAbs are affected by factors such as affinity, dose, patient variability, and cachexia. and further influenced by tumor-extrinsic factors such as the quality of life and the development of anti-therapeutic antibodies (She ng et al., 2017). Existing PK / PD prediction models are ineffective in predicting optimal doses. It relies on in vitro and PBMC-based assays (Deng et al., 2016). While the presently disclosed subject matter uses PET to non-invasively detect tumors in real time, We demonstrate that PD-L1 occupancy by therapeutic antibodies can be measured.

[0137] such as atezolizumab, supported by peripheral pharmacodynamic evaluation and PK / PD modeling Radiolabeled antibodies were used to determine the mAb dose required to achieve the desired PD-L1 occupancy in tumors. It is routinely used to predict plasma levels of antibodies (Deng et al., 2016). Intracellular and tumor concentrations are influenced by antibody isotype and biophysical properties such as charge and valency. Because these measurements and mathematical modeling-derived occupancy predictions are often subject to the This is specific to this antibody and therefore does not generalize such observations to other PD-L1 mAbs. The number of PD-L1 treatments available is expanding. Evaluate therapeutic mAbs for antibody kinetics and tumor target engagement There is a need for tools that can be used to do this. The subject matter of this disclosure addresses this need. Combined with in vitro and in vivo data using WL12-PET, Combined in silico modeling studies defined PD-L1 saturation / occupancy in tumors. demonstrated that PD-L1 can be quantified for all therapeutic mAbs in clinical trials. This is a concept that can be applied to:

[0138] Taken together, the data disclosed herein demonstrate the dynamic changes in PD-L1 expression in tumors and in response to treatment. PD-L1 saturation / occupancy by therapeutic antibodies is independent of two features: antibody specificity and by considering tumor intrinsic and extrinsic parameters, Three different therapeutic antibodies, AtzMab, AveMab, For DurMab, the results of the present disclosure relating dose to PD-L1 occupancy in tumors , which is expected to be related to treatment response and medication effect.

[0139] 3.3.7. Overview. The subject matter of the present disclosure is a method for detecting and treating variable and dynamic PD-L1 expression levels using radiolabeled PD-L1 binding peptides. The level can be detected noninvasively, and tumor-intrinsic parameters (PD-L1 expression, recurrence) can be analyzed. , interstitial pressure) and extrinsic parameters (antibody isotype, kinetics, ATA, catabolism) However, it can be used to measure tumor occupancy and therefore PD-L1 expression in tumors. : Providing a universal tool for monitoring the therapeutic activity of PD-1 interaction-blocking PD-L1 antibodies Demonstrate that it provides

[0140] [ 64 Cu]WL12 study demonstrated variable and dynamic changes in PD-L1 expression can be quantified within standard clinical workflow and is useful for patient selection and monitoring treatment Demonstrate significant clinical significance.

[0141] Existing PK / PD prediction models for antibodies rely on in vivo data to predict optimal doses. However, it relies on in vitro and PBMC-based assays (Deng et al., 2016). However, the subject matter of the present disclosure uses PET to non-invasively detect therapeutic tumors in real time. We demonstrate that PD-L1 occupancy by antibodies can be measured.

[0142] Antibody kinetics and tumor response in the ever-expanding array of PD-L1 therapeutic mAbs There is a need for tools that can be used to assess target engagement. The subject matter of the present disclosure addresses this need. In silico modeling studies combined with in vitro and in vivo data demonstrated that it is possible to quantify PD-L1 saturation / occupancy in tumors, which has been shown to be useful in clinical trials. This concept is applicable to all PD-L1 therapeutic mAbs in clinical trials.

[0143] (References) All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All publications mentioned in this specification are indicative of the level of ordinary skill in the art to which the disclosed subject matter pertains. , patent applications, patents, and other references (e.g., websites, databases, etc.) , the entire contents of which are hereby incorporated by reference as if each individual publication, patent application, patent, and other reference were incorporated by reference. references to the same extent as if the documents were specifically and individually indicated to be incorporated by reference. Numerous patent applications, patents, and other references are incorporated herein by reference. Although references to any of these documents are made to the present invention, such references are not intended to be limiting unless expressly stated to the extent that they are incorporated herein by reference. It will be understood that no admission is made that any of the information contained herein forms part of the general knowledge of the public. In the event of a conflict between the specification and any incorporated references, the specification (including any amendments thereto) shall govern. This document (including the preceding paragraphs, which may be based on incorporated references) shall take precedence. The specification uses standard, art-accepted meanings of terms unless otherwise indicated. Standard abbreviations for various terms are used herein. Agoram, BM (2009) Use of pharmacok inetic / pharmacodynamic modeling for st arting dose selection in first-in-human t Br J Clin Pharmacol 67, 153-160. Anderson, C.J., and Ferdani, R. 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(2 015) Structure of the Complex of Human P rogrammed Death 1, PD-1, and Its Ligand PD-L1. Structure 23, 2341-8. The foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding. However, those skilled in the art will recognize that certain changes and modifications can be practiced within the scope of the appended claims. This will be understood by others. Sequence Listing: SEQ ID NO: 1 WL12 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-L eu-Hyp-Trp-Ser-Trp(methyl)-NMeNle-NMeNle-Lys-Cy s-)-Gly-NH2) SEQ ID NO: 2 DK-A-221 amino acid sequence = cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-H is-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMe Nle-Lys-Cys-)-Gly-NH2

Claims

1. Peptides with binding specificity for programmed death ligand 1 (PD-L1) and reporter and optionally a linker, wherein the linker is a conjugate of the imaging agent. A linker, if present, links the peptide to the reporting moiety and If no anchor is present, the reporting site is located at the first amino acid of the peptide. The imaging agent is directly attached to the peptide via a secondary amine.

2. the peptide having binding specificity for PD-L1 comprises: amino acid Y56 of PD-L1; 10. The imaging agent of claim 1, which interacts with E58, A113, M115, and Y123. 。

3. the peptide having binding specificity for programmed death-ligand 1 (PD-L1), At least 80% of peptide WL12, DK-A-221, or DK-A-222 3. The imaging agent of claim 1 or 2, having sequence identity.

4. the peptide having binding specificity for programmed death-ligand 1 (PD-L1), At least 85% of peptide WL12, DK-A-221, or DK-A-222 The imaging agent of claim 3 having sequence identity.

5. The peptide has binding specificity for a programmed death-ligand 1 (PD-L1) imaging agent. The peptides have at least 9 5. The imaging agent of claim 4, having 0% sequence identity.

6. the peptide having binding specificity for programmed death-ligand 1 (PD-L1), 100% sequence identity to peptide WL12, DK-A-221, or DK-A-222 The contrast agent of claim 5 .

7. The reporting moiety may be a chelator, a radiolabeled substrate, a fluorescent dye, a photoacoustic reporter 10. The method of claim 1, wherein the Raman-active reporting molecule is selected from the group consisting of: a Raman-active reporting molecule; The contrast agent according to claim 1.

8. the reporting moiety is a chelating agent, the chelating agent being selected from the group consisting of: The contrast agent of claim 7, selected from: DOTAGA (1,4,7,10-tetraazabicyclo[4.1.2.1]). Clododecane, 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 1,4,7,10-tetraaza-cyclododeca-1-yl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododeca-1- 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl)-acetic acid)), 3p-C-DEPA (2-[(carboxymethyl)][5-(4-nitrophenyl)-acetic acid)), Nyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazaniline [chlorododecan-1-yl]pentan-2-yl)amino]acetic acid), TCMC (2-(4-isopropyl Thiocyanobenzyl)-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-tetraazacyclododeca 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-TE1A 1P (4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methacrylonitrile) tetracarboxylic acid), CB-TE2P (1,4,8,11-tetraazacyclotetradecane- 1,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetraacetic acid) 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) Acid), (NOTAGA) 1,4,7-triazonane-1,4-diyl)diacetic acid DFO (des Ferroxamine), 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] (6,6,6) eicosane, 3,6,10,13,16,19-hexaazabicyclo[ 6.6.6]eicosane-1,8-diamine), Sarar (1-N-(4-aminobenzyl )-3,6,10,13,16,19-hexaazabicyclo[6.6.6] eicosane- 1,8-diamine), AmBaSar (4-((8-amino-3,6,10,13,16,1 9-hexaazabicyclo[6.6.6]icosan-1-ylamino)methyl)benzoic acid), and BaBaSar.

9. 8. The imaging agent of claim 7, wherein the chelating agent is selected from the group consisting of: 【Chemical 1】

10. the reporting moiety is a chelating agent, the chelating agent comprising: ９４ｍ Tc, ９９ｍ Tc、 １１１ In、 ６７ Ga、 ６８ Ga、 ８６ Y、 ９０ Y、 １７７ Lu、 １８６ Re、 １ ８８ Re, ６０ Cu, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ５５ Yes, ５７ Yes, ４７ Sc ２２５ Ac ２１３ Bi ２１２ Bi ２１２ Pb １５３ S １６６ Ho , １５２ Gd, ８２ Rb, ８９ Zr, and １６６ Dy The contrast agent of claim 7 further comprising a metal.

11. the reporting moiety is a radiolabeled substrate, the radiolabeled substrate comprising: １１ C. １ ３ N、 １５ O、 １２３ I、 １２４ I、 １２５ I、 １２６ I、 １３１ I、 ７５ Br、 ７６ B r, ７７ Br, ８０ Br, ８０ｍ Br, ８２ Br, ８３ Br, １８ F, and ２１１ At 8. The imaging agent of claim 7, comprising a radioisotope selected from the group consisting of:

12. 12. The imaging agent of claim 11, wherein the radiolabeled substrate comprises an 18F-labeled substrate.

13. The 18F-labeled substrate is 2-fluoro-PABA, 3-fluoro-PABA, 2-fluoro- mannitol, and N-succinimidyl-4-fluorobenzoate The contrast agent of claim 12 , wherein

14. The method of claim 1, wherein the reporting moiety is directly incorporated into the peptide. Contrast agent.

15. 15. The method of claim 14, wherein the reporting moiety comprises a radiolabeled amino acid of the peptide. The contrast agent described herein.

16. The radiolabeled amino acid is selected from the group consisting of iodotyrosine and fluorotyrosine. The contrast agent of claim 15, wherein

17. the reporting moiety is a fluorescent dye, the fluorescent dye being selected from the group consisting of: The contrast agent according to claim 7, which is a carbocyanine, an indocarbocyanine, an oxacary Bosyanine, tsuicarbocyanine, merocyanine, polymethine, coumarin, rhodamine, Xanthene, fluorescein, boron-dipyrromethane (BODIPY) dyes, Cy5, C y5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag -S750, AlexaFluor660, AlexaFluor680, AlexaF luor700, AlexaFluor750, AlexaFluor790, Dy67 7, Dy676, Dy682, Dy752, Dy780, DyLight547, Dyl light647, HiLyte Fluor 647, HiLyte Fluor 68 0, HiLyte Fluor 750, IR Dye 800, IRDye 800C W, IRDye 800RS, IRDye 700DX, ADS780WS, ADS83 0WS, and ADS832WS.

18. The reporting moiety is a photoacoustic reporting molecule, and the photoacoustic reporting 8. The imaging agent of claim 7, wherein the binding molecule is selected from the group consisting of a dye or a nanoparticle.

19. 20. The contrast agent of claim 18, wherein the dye comprises a fluorescent dye.

20. The fluorescent dye is indocyanine green (ICG), Alexa Fluor 750 , Evans Blue, BHQ 3, QXL 680, IRDye 880CW, MMPS ense 680, methylene blue, PPCy-C8, and Cypate-C18 20. The imaging agent of claim 19, selected from the group consisting of:

21. 19. The contrast agent of claim 18, wherein the nanoparticles are selected from the group consisting of: plasma Sonic nanoparticles, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles Particles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd ２ O ３ Nanoparticles, single Multi-walled carbon nanotubes, dye-loaded perfluorocarbon nanoparticles, and superparamagnetic oxides Iron nanoparticles.

22. The reporting moiety is a Raman-active reporting molecule, The coating molecules are single-walled carbon nanotubes (SWNTs) and surface-enhanced Raman scattering (S 8. The imaging agent of claim 7, selected from the group consisting of phosphodiesterase inhibitors (PSE) agents.

23. the SERS agent comprises a metal nanoparticle labeled with a Raman-active reporter molecule. Item 23. The contrast agent according to Item 22.

24. 24. The imaging agent of claim 23, wherein the Raman-active reporter molecule comprises a fluorescent dye.

25. The fluorescent dye is selected from Cy3, Cy5, rhodamine, and chalcogenopyrylium dyes.

25. The imaging agent of claim 24, selected from the group consisting of:

26. 8. The imaging agent of claim 7, wherein the linker is selected from the group consisting of: (a) 【Chemistry 2】 (In the formula: Rpt is the reporting site; W １ is C １ -C ６ Alkylene, C ３ -C ６ Cycloalkylene and arylene selected from the group consisting of: 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 is, independently, H or C １ -C ４ is alkyl; Each R ２ are independently H or -COOR ３ where each R ３ independently , H., C. １ -C ６ Alkyl, C ２ -C １２ Aryl or C ４ -C １６ Alkyl aryl be; 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. There is; 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 F Tetraalkynyl, C １ -C ８ alkoxy, or a bond, each of which may be 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 -C O-、-NR Ｂ -CO ２ -、-CO-NH-、-CO ２ -EH-、-EEER Ｂ -、-CO ２ -N Ｂ - , or a 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 heterocyclic optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkyl alkoxy, optionally substituted mono- or dialkylamino, optionally substituted alkyl thio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted mono- or dialkylcarboxamide, optionally substituted aryl, or optionally substituted heteroaryl; R Ｂ is optionally replaced for each occurrence of optionally substituted alkyl, optionally substituted alkoxy, optionally substituted mono- or di-alkyl aryl, optionally substituted alkylthio, optionally substituted aryl, or optionally substituted aryl substituted heteroaryl); or (c) Amino acid linker.

27. The imaging agent is selected from the group consisting of compounds of formula (I), formula (II), or formula (III). The contrast agent of claim 1 selected from: 【Chemistry 3】 DK-A-221-(L) ｎ -Rpt(II); or 40-2-222-()) ｎ -2pt(999); (In the formula: n is an integer selected from the group consisting of 0 and 1; L is a linker; and Rpt is the reporting site; and wherein the reporting moiety or linker, if present, is of formula (I), formula (II), or attached to a primary amine group of an amino acid of said peptide comprising said imaging agent of formula (III) (I am doing it).

28. The linker, if present, is １３ Ornithine (Orn) Primary A 28. The imaging agent of claim 27, wherein the amine group is bonded to the amine group.

29. 28. The imaging agent of claim 27, wherein the reporting moiety comprises a DOTAGA chelator. 。

30. The DOTAGA chelating agent is ６４ 30. The method of claim 29, further comprising Cu radiometal. Contrast agent.

31. 28. The imaging agent of claim 27, wherein the compound of formula (I) is: 【Chemistry 4】

32. 1. An imaging method for detecting programmed death-ligand 1 (PD-L1), comprising the steps of: How to: (a) providing an effective amount of an imaging agent according to any one of claims 1 to 27; (b) contacting one or more cells or tissues with said imaging agent; (c) Creating an image and detecting PD-L1.

33. contacting the one or more cells or tissues with the imaging agent in vitro; The imaging method of claim 32, which is performed in vivo or ex vivo. Law.

34. The contacting of the one or more cells or tissues with the imaging agent is performed in a subject.

34. The imaging method of claim 33,

35. The subject may be a human, a rat, a mouse, a cat, a dog, a horse, a sheep, a cow, a monkey, a chicken, or the like.

35. The imaging method of claim 34, wherein the animal is a mammal, such as a cat, a mammal, or an amphibian.

36. the detection of PD-L1 is within about 60 to 120 minutes after administration of the imaging agent to the subject.

33. The imaging method of claim 32, wherein

37. The imaging method of claim 32, wherein the imaging method is used to detect cancer. aging method.

38. 38. The imaging agent of claim 37, wherein the cancer is selected from the group consisting of: blastoma, carcinoma , glioma, leukemia, lymphoma, melanoma, myeloma, sarcoma, head and neck cancer, head and neck Lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, esophageal cancer, stomach cancer Cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer ductal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, brain tumors, adenomas, and metastatic cancer 。

39. 33. The imaging method of claim 32, wherein the imaging method is used to detect solid tumors. Amazing method.

40. The solid tumor is selected from the group consisting of brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovary, and uterine tumor. Located in an organ selected from the group consisting of the cervix, stomach, rectum, bladder, uterus, testes, and pancreas 40. The imaging method of claim 39.

41. 33. The imaging method of claim 32, wherein the imaging method is used to detect infectious diseases. aging method.

42. 42. The imaging method of claim 41, wherein the infection is a microbial infection.

43. The microbial infection is Mycobacterium tuberculosis, Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus and coagulase Negative Staphylococci including Staphylococcus aureus, Enterococcus, Streptococcus pneumoniae, influenza Enzyme, Bacteroides, Acinetobacter, Helicobacter, Candida, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus An infectious disease caused by one or more microorganisms selected from the group consisting of facium (VRE)? The imaging method according to claim 42, selected from the group consisting of?

44. The imaging method according to claim 32, wherein the imaging method is used to detect inflammation? Method.

45. The inflammation is asthma, autoimmune disease, autoinflammatory disease, celiac disease, diverticulitis, glomerulonephritis? Inflammatory hidradenitis, allergy, inflammatory bowel disease, interstitial cystitis, otitis media, pelvic inflammatory disease? Reflux injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, lupus, systemic lupus erythematosus? And the contrast agent according to claim 44, which is related to a disorder selected from the group consisting of vasculitis? Method.

46. The imaging method according to claim 45, wherein the inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus? Method.

47. The imaging method according to claim 32, wherein the imaging method is used to detect one or more immune cells in a tumor? Method.

48. The imaging method according to claim 32, wherein the imaging method is used to detect the systemic distribution of immune cells within a tumor or in a subject? Method.

49. The imaging method according to claim 32, wherein the imaging method is used to detect an immune cell response to an infectious disease? Method.

50. The imaging method according to claim 32, wherein the imaging method is used to detect an intratumoral or normal tissue response of immune cells to an inflammatory disease? Method.

51. The imaging method according to claim 32, wherein the imaging method detects the PD-L1 expression level in the subject? Method.

52. The imaging method according to claim 32, wherein the imaging method measures the occupancy or target engagement of PD-L1 in a tumor site or normal tissue of the subject by an antibody or a peptide or a low molecular weight agent? Method.

53. A kit for detecting programmed death ligand 1 (PD-L1), comprising the contrast agent according to any one of claims 1 to 27? ​ ​

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