Tumor and immune cell imaging based on PD-L1 expression

Abstract

Provided are imaging agents capable of detecting programmed death-ligand 1 (PD-L1), compositions, kits, and methods comprising the imaging agents. [Solution] The imaging agent comprises a conjugate of a peptide having binding specificity for programmed death-ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, wherein the linker, if present, connects the peptide and the reporting moiety, and when the linker is not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is incorporated by reference in its entirety herein. 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 with funding from the National Institutes of Health (NIH) awarded NIH R01CA1663. This invention was made with Government support under Grant No. 10-143444. 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 will guide immunotherapy strategies to enhance immune-modulatory therapy. There are few contrast agents that can be used.

[0004] Immunotherapy, which suppresses one's own immune system to kill cancer cells, is a central treatment for various cancers. (Topalian et al., 2016) has shown a significant improvement in treatment outcomes. Despite these results, many cancers do not respond to immunomodulatory therapies. Existing companion diagnostics that act via tumor immunology provide only a snapshot of the dynamic tumor immune environment. and often do not accurately predict treatment response (Mansfield and Do Noninvasive imaging techniques provide quantitative, real-time insight into tumor biology. can provide assessment and guide drug development (Willmann et al., 2008).

[0005] Positron emission tomography (PET), the most molecular and quantitative translational imaging technique, is 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 used to assess 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 other imaging methodologies, including Raman imaging, are relevant for immunomodulatory therapies. It can provide rapid, real-time assessment of target expression in ongoing clinical trials. It can bring big benefits.

[0006] Programmed death-ligand 1 (PD-L1) is an immune-regulated protein 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 of 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 subject matter of the present disclosure relates to a method for treating cancer, comprising administering to a patient a therapeutically effective amount of a PD-L1 inhibitor against programmed death-ligand 1 (PD-L1). A conjugate of a peptide having binding specificity for a reporting moiety, and optionally The imaging agent includes a linker, which, if present, is a linker between the peptide and the reporter. In the absence of a linker, the reporting portion is linked to the peptide. The imaging agent is directly attached to the peptide via the primary amine of the amino acid. In some embodiments, the reporting moiety is incorporated directly into the peptide, e.g., The binding moiety is a radiolabel of the peptide, such as a 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 of formula (III): [ka] DK-A-221-(L) n -Rpt (II); or DK-A-222-(L) n -Rpt (III); where 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 pentaerythrocyte containing an imaging agent of formula (I), formula (II), or formula (III). (attached to the primary amine group of an amino acid of the peptide).

[0010] In a particular embodiment, the compound of formula (I) is WL12 DOTA: [ka]

[0011] In another aspect, the subject matter of the present disclosure provides a method for the treatment of a patient infected with programmed death-ligand 1 (PD-1), comprising the steps of: Imaging methods for detecting: (a) 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, is In the absence of a linker, the reporting portion is not linked to the peptide. 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) generating an image to detect PD-L1. is a compound of formula (I), or Y56, E58, A113, M11 of PD-L1 5 and a peptide that interacts with Y123.

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

[0013] In yet a further aspect, the subject matter of the present disclosure relates to a method for treating cancer by inhibiting programmed death-ligand 1 (PD-L1). The present invention provides a kit for detecting a PD-L1-specific antibody against a PD-L1 ligand. A conjugate of a peptide having binding specificity for a reporting moiety, and optionally The imaging agent may further comprise a linker, if present, which may be a linker between the peptide and the reporter molecule. When the linker is not present, the reporting portion is linked to the peptide. The present invention includes an imaging agent that is directly attached to a peptide via the primary amine of an amino acid of the

[0014] Certain aspects of the presently disclosed subject matter are described above and are generally defined by the presently disclosed subject matter. The present invention is best described in detail in the accompanying drawings, which are taken in part or in part as being best explained herein below. Other aspects will become apparent as the description proceeds with reference to the examples and drawings. [Brief description 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 1 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 in the groove of PD-L1. WL12 is shown in cyan. A surface diagram of PD-L1 is shown in grey, with ribbons and major side chains in magenta; Figure 1C shows that WL12 mimics the binding of PD-1 to PD-L1. The structure of PD-1 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; [Diagram 2] FIG. 1 shows the far-UV CD spectrum of peptide WL12. [Diagram 3] Figure 1 shows 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; [Diagram 5] 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 3 shows RP-HPLC purification of [PDL1-PD-Cu2+]; [Figure 7] 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 PD-L1 binding peptide WL12; Figure 8A shows a competitive inhibition assay demonstrating the affinity of the WL12 analog to inhibit PD-1:PD-L1 interaction; Figure 8B shows flow cytometry histograms of cell lines used in the in vitro study that show 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] Representative curves 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 2 shows RP-HPLC chromatograms of [64Cu]WL12 radioactive tracer (red) and the "non-radioactive" WL12-Cu2+ complex; [Figure 12] Figure 1 shows RP-HPLC chromatograms of a mixture of PDL1-PD and [PDL1-PD-Cu2+]; [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 administered 150 μCi of [64Cu]WL12 intravenously and images were acquired at 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 demonstrates strong immunoreactivity (brown) in hPD-L1 tumors; [Figure 16] Figure 1. 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 to CHO (blue arrow) tumors confirms PD-L1-mediated uptake of the radiotracer; [Figure 17] Figure 1 shows ex vivo biodistribution analysis 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 min after injection. For blocking studies, mice were administered an excess of peptide (pep) with 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: Evaluation of WL12-IR800CW in CHO and hPDL1 tumor-bearing mice: (Fig. 19A) Representative images of mice and ex vivo organs injected with 5 nmoles of WL12-IR800CW recorded 24 hours after conjugate injection, (Fig. 19B, blocking) Representative images of mice injected with 25 nmoles of unmodified WL12 and 5 nmoles of WL12-IR800CW acquired at 24 h pi; (Fig. 19C) Quantification of ex vivo biodistribution of WL12-IR800CW in selected organs and tumors from mice treated with 1 nmole, 3 nmole, and 5 nmoles of conjugate and blocked with WL12 (numbers indicate corresponding organs, n=4); [Figure 20] Figure 1: [111In]AtzMab 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: [64Cu]WL12-PET detects AtzMab accumulation in tumors; (A) Whole-body [64Cu]WL12 images show specific accumulation of radioactivity to hPD-L1 tumors by 60 min pi; (B) [64Cu]WL12 uptake is significantly decreased in hPD-L1 tumors in mice administered a 20 mg / Kg dose of AtzMab 24 h prior to tracer injection; (C) Corresponding biodistribution studies confirmed the feasibility of [64Cu]WL12 to detect AtzMab PD-L1 engagement to tumors; (D) WL12 inhibits AtzMab binding to PD-L1. hPD-L1 cells incubated with serial dilutions of WL12 were stained with Cy5-AtzMab or commercial 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] [64Cu]WL12-PET detects accumulation of AtzMab in triple-negative breast cancer xenografts; [64Cu]WL12 uptake is significantly reduced in MDAMB231 tumors in mice administered a 20mg / Kg dose of AtzMab 24 hours prior to tracer injection; [Diagram 23] Figure 1 shows: (A) Structure of Wl12-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: Evaluation of WL12-IR800 in CHO and hPDL1 tumor-bearing mice; (A) Representative images of mice injected with 5 nmol WL12-IR800 and ex vivo organs recorded 24 h after conjugate injection; (B, blocking) Representative images of mice injected with 25 nmol unmodified WL12 and 5 nmol WL12-IR800 acquired 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 conjugate and blocked with WL12 (numbers indicate corresponding organs, n=4); [Diagram 25] 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) confirming 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 2. 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 arrows, high PD-L1 expression) and CHO (blue arrows, low PD-L1 expression) tumors (n=3) confirm PD-L1-mediated uptake of the radiotracer; [Figure 27] Figure 1: MDAMB231 and SUM149 tumor-bearing mice were injected with a 20mg / Kg dose of Atezolizumab; 20 hours after mAb administration, mice were injected with 20μCi 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 decreases 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 1 or 10 mg / Kg doses. 24 or 120 hours after mAb administration, mice were injected with [64Cu]WL12 and tumor accumulation of radioactivity was measured by biodistribution studies. As expected, complete blockade of PD-L1 was observed at both 24 and 120 hours at the 10 mg / Kg dose. At the 1 mg / kg dose, increased accumulation of [64Cu]WL12 was seen 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 may be used to analyze PD-L1 therapeutic mAb residence time in tumors; [Diagram 30] 1 shows the chemical structures of DK-A-221 and DK-A-222; [Diagram 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 show excellent pharmacokinetics of [64Cu]DK222; [Diagram 32] Figure 1 shows the biodistribution of [64Cu]DK222 in NSG mice bearing CHO / CHO-hPD-L1 tumors; [Diagram 33]Figure 33 demonstrates 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 that of PD-1 to AtzMab (red and cyan), AveMab (orange and cyan) and DurMab (blue and cyan). Non-interacting residues are shown in grey. The diverse contacts encompassing the common binding region (cyan) explain the diverse binding mechanisms of different therapeutic mAbs. Figure 33B shows that WL12 inhibits AtzMab, AveMab and DurMab conjugated to Cy-5 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 PBS control. [64Cu]WL12 binding in PD-L1 negative CHO and SUM149 cells is also shown. ****, P<0.0001; NS, non-significant; [Diagram 34](Fig. 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 grey. 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; (Fig. 34B) WL12 inhibits binding of Cy5-conjugated PD-1-Fc protein to PD-L1 in hPD-L1 cells. Mean fluorescence intensity determined by flow cytometry; (Fig. 35C) WL12 (5 nM) inhibits 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; [Diagram 35] Figure 35 demonstrates that PD-L1 engagement by PD-L1 mAb 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 (Figure 35A,B), HCC827 (Figure 35C,D), and hPD-L1 / CHO (Figure 35G,H) 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 (Figure 35A,D,G) and ex vivo biodistribution (Figure 35B,E,H). Figure 35C, Figure 35F and Figure 35I show IHC staining for PD-L1 from corresponding tumors. ****, P<0.0001. ***, P<0.001; NS, non-significant; [Diagram 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 of tumor PD-L1 expression detected with [64Cu]WL12 and its engagement by AtzMab. Figure 37A shows the increase of 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 (5nM) inhibits the binding of AtzMab, AveMab and DurMab (2nM) conjugated to Cy5 to A549-iPD-L1 cells treated with doxycycline for 72 hours. Figure 37C shows that [64Cu]WL12 binding to A549-iPDL1 cells (doxycycline for 72 hours) is significantly decreased in the presence of 60nM AtzMab compared to the control. Figure 37D and Figure 37E show that [64Cu]WL12 uptake in A549-iPDL1 xenografts is significantly lower in mice that received intravenous AtzMab 24 hours prior to radiotracer injection compared to 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 of 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 20mg / Kg AtzMab 24 hours prior to radiotracer injection. ****, P<0.0001; NS, non-significant; [Figure 39]Figure 39A-E demonstrates tumor PD-L1 engagement by three different PD-L1 therapeutic mAbs quantified with [64Cu]WL12. [64Cu]WL12 uptake 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; [Diagram 40] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in MDAMB231-bearing mice treated with AtzMab (20mg / Kg), AveMab (10mg / Kg), or DurMab (10mg / Kg) 24 hours before radiotracer injection. ****, P<0.0001; NS, non-significant; [Diagram 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 depicting 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 MDAMB231 tumor-bearing mice administered 0.06mg / kg, 0.6mg / kg and 3.2mg / kg AtzMab (Figure 41A). Figure 41B and Figure 41C show ex vivo quantification of [64Cu]WL12 uptake in tumors of mice treated with increasing doses of AtzMab (0.0009-24mg / kg). AtzMab was injected 24 hours prior to 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. Figure 41D and Figure 41E show the AtzMab (mg / kg) dose effect on tumor PD-L1 occupancy over time, showing an increase in free PD-L1 ligand at 0.6 or 1 mg / kg doses of AtzMab, but not at 10 or 20 mg / kg AtzMab doses, 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; [Diagram 42] Figure 1 shows the ex vivo biodistribution of [64Cu]WL12 in MDAMB231 tumor-bearing mice at increasing doses of AtzMab (0.0009-12mg / Kg) 24 hours before tracer injection; [Diagram 43] 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 PREFERRED EMBODIMENTS

[0017] The subject matter of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings, in which: Some, but not all, embodiments of the illustrated subject matter are shown. The subject matter of the present disclosure may be embodied in many different forms, and the same elements may be used interchangeably herein. The present invention should not be construed as being limited to the embodiments set forth in the specification; rather, these embodiments are 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 of skill in the art to which the presently disclosed subject matter pertains. This invention will come to the mind of 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 Imaging Agents In some embodiments, the subject matter of the present disclosure relates to an immune checkpoint marker, 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 characterize tumor PD-L1 expression. and can be detected immediately after administration to a subject.

[0019] Thus, in some embodiments, the subject matter of the present disclosure relates to a method for treating programmed death ligand 1 (P Conjugation of a peptide with binding specificity for the IL-1 domain and a reporting moiety. and optionally a linker, wherein the linker, if present, is In the absence of a linker, the reporting moiety is not linked to the 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 comprises radiolabeled amino acids.

[0020] In some embodiments, the binding specificity is 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 that has 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 eNle-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 mutant that mutates amino acids Y56, E58, D61, and In some embodiments, WL12 can interact with PD-L1 5 and A113. In a specific embodiment, WL12 can interact with one amino acid of PD-L1. It can interact with acids Y56, E58, A113, M115 and Y123. So, the peptide that interacts with PD-L1 is DK-A-221. Peptide DK-A-2 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 binds to the five amino acids of PD-L1. In a specific embodiment, DK-A-221 is capable of interacting 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 binds to the 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 is capable of interacting 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: 1. Binding specificity to PD-L1. A peptide having the sequence of SEQ ID NO:2 may have at least 80% sequence identity to SEQ ID NO:2. A peptide having binding specificity for D-L1 has a binding specificity of at least 85% to SEQ ID NO:1. The peptide 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 peptide with binding specificity for PD-L1 may have sequence identity to SEQ ID NO:1. The antibody 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. A 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 nucleic acid or between two or more polynucleotide sequences is determined by comparing the sequences. In the art, "identity" is sometimes determined by A polypeptide or polynucleotide is a polypeptide or polynucleotide that is a polypeptide of interest, 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 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 to 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. Amplification and percent identity calculations were performed using LASERGENE Bioinformatics Megalign program of computing suite (DNASTAR, Maddie Multiple alignment of sequences can be performed using the GABA 10001 ... The Clustal alignment method (Higgins and Sharp (198 9) CABIOS. 5:151-153) was used 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 a non-naturally occurring amino acid that is chemically similar to acid.

[0024] The terms "naturally occurring amino acid" and "naturally encoded amino acid" are used interchangeably. The amino acids that can be used and are 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-carbo 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 that are capable of binding to acetylcholine, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs include those with modified R groups, e.g. norleucine) or a modified peptide backbone, 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 not found in translation complexes. "Naturally occurring arylsulfates" refers to compounds that are not incorporated into a growing polypeptide chain by "Amino acids" are naturally encoded amino acids, including the 20 standard amino acids, These are amino acids that arise by modification (e.g., post-translational modification) of the Amino acids that are not naturally incorporated into the growing polypeptide chain 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 replace 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 homocitrulline (NMcCit), N-methyl homocitrulline (N-MeHoCit), 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 (nitrilophenylalanine ophe), aminophenylalanine (aminophe or amino-Phe), Diphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-car boxyglu), hydroxyproline, 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-hydro Proxyproline (Hyp). γ-Carboxyglutamate, ε-N,N,N-trimethyl Lysine, -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine arginine, 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. 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, Addition of chemicals such as nesyl groups, isofarnesyl groups, sulfoxide groups, and fatty acid groups, They may be modified, such as by 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 ends, 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 child 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) Lazacyclododecane, 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] Chloro[5.5.2]tetradecane, DEPA (7-[2-(bis-carboxymethylamino) (No)-Ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraazacyclo dodec-1-yl-acetic acid), 3p-C-DEPA(2-[(carboxymethyl)][5-(4- Nitrophenyl-1-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetra tetraazacyclododecan-1-yl]pentan-2-yl)amino]acetic acid), TCMC(2 -(4-isothiocyanobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra tra-(2-carbamonylmethyl)-cyclododecane), oxo-DO3A (1-oxa-4, 7,10-Triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,1 0-triacetic acid), p-NH 2 -Bn-Oxo-DO3A (1-Oxa-4,7,10-tetraazacyl 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-tetraazacyl Clotetradecane-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-glutar (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-tris(2-mercaptoethyl) cyclononane), diamsal (1,8-diamino-3,6,10,13,16,19-hexafluorophenyl) 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 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, published November 20, 2014, to Pomper et al. Homopolyvalent and heteropolyvalent inhibitors of prostate specific membrane antigen (Pmsa) and their uses. No. 6,399,433, 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 compound further comprises a radioactive metal selected from the group consisting of:

[0033] In other embodiments of the imaging agent 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, oxacane Rubocyanine, tsuicarbocyanine, merocyanine, polymethine, coumarin, rhodamine , xanthene, fluorescein, boron-dipyrromethane (BODIPY) dyes, or Derivatives of BODIPY, BODIPY FL, BODIPY R6G, BODIPY TR, 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-cyclohexadiene-1-ylidene Ammonium Perchlorate), IRDye 800CW, IRDye 800RS, IR Dye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0035] In other embodiments of the imaging agent of the present disclosure, the reporting moiety is a photoacoustic reporting molecule. and the photoacoustic reporting molecule is selected from the group consisting of a dye or a nanoparticle. In certain embodiments, the dye comprises a fluorescent dye. In 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 nanoparticles, Plasma nanoparticles, including but not limited to nanocage, gold nanostars, and gold nanoclusters. Zumonic nanoparticles, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles nanoparticles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd 2 O 3 Nanoparticles, Single-walled carbon nanotubes, dye-loaded perfluorocarbon nanoparticles, and superparamagnetic acids The nanoparticles are selected from the group consisting of iron oxide nanoparticles.

[0037] In other embodiments of the imaging agent 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. SERS agents consist of metals (e.g., gold or silver) labeled with Raman-active reporter molecules. In a more specific embodiment, 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 the reporting site; W 1 is C 1 -C 6 Alkylene, C 3 -C 6 cycloalkylene, and arylene; W 2 -NR 1 -(C =O)-, -NR 1 -(C=S)-, -(C=O)-NR 1 -, -(C=S)-NR 1 -and- S-, where each R 1 are independently H or C 1 -C 4 With alkyl Each R 2 are independently H or -COOR 3 where each R 3 is, independently, H, C 1 -C 6 Alkyl, C 2 -C 12 Aryl or C 4 -C 16 Alkyl aryl 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 attachment point between the linker and the peptide); (b) Rpt-XYZW 3 - wherein Rpt is a reporting site; X and Z are each independently C 1 - C 8 Alkyl, C 2 -C 8 Alkenyl, C 2 -C 8 Alkynyl, C1 -C 8 Heteroalkyl , C 2 -C 8 Heteroalkenyl, C 2 -C 8 Heteroalkynyl, C 1 -C 8 Alkoxy, too or a bond, each of which may be 0 to 5 R A may be substituted with; Y and W 3 teeth, Independently, -O-, -S(O) p -, -NH-, -NR B -, -CH=CH-, -CR B = CH-, -CH=CR B -, -NH-CO-, -NH-CO 2 -, -NR B -CO-, -NR B -CO 2 -, -CO-NH-, -CO 2 -NH-, -CO-NR B -, -CO 2 -NR B - or a combination p is 0, 1, or 2; R A Each occurrence of is a halogen, hydroxyl , amino, cyano, nitro, CO 2 H, optionally substituted alkyl, optionally substituted silyl cycloalkyl, optionally substituted heterocycle, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted mono- or di-alkynyl optionally substituted alkylamino, optionally substituted alkylthio, optionally substituted alkylsulfinyl, optionally substituted alkylsulfonyl, optionally substituted mono- or dialkylcarboxy amide, optionally substituted aryl, or optionally substituted heteroaryl; R BEach occurrence of is an optionally substituted alkyl, an optionally substituted alkoxy, an optionally substituted aryl ... optionally substituted mono- or dialkylamino, optionally substituted alkylthio, 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): A compound selected from the group: [ka] DK-A-221-(L) n -Rpt(II); or DK-A-222-(L) n -Rpt(III); where 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 pentaerythrocyte 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 is 6 4 It further contains Cu radiometal.

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

[0042] Those of skill in the art, upon consideration of the subject matter of this disclosure, will recognize key It will be appreciated that various rate agent / radiometal ion combinations will be suitable. Representative chelating agents are known in the art. Non-limiting examples include The chelating agents and linkers are described in U.S. Patent Application Publication No. 2015 / 0246144 and No. 2015 / 0104387, each of which is incorporated by reference in its entirety. and is hereby incorporated by reference.

[0043] In some embodiments, the imaging agent is In some embodiments, the imaging agent can detect PD-L1 in vivo or ex vivo. PD-L1 can be detected in vivo. PD-L1 is expressed by various tumors. Its overexpression is thought to be 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 agent of the present disclosure that inhibits interaction with plasma membrane 1 (PD-1) 50 is about 100nM~ In some embodiments, the IC 50 is less than 100 nM, other implementations in other 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 qualitatively described as "strong," "weak," "high," or 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 subject matter of the present disclosure relates to an immune checkpoint marker, 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 the preparation of a programmed death ligand comprising the steps of: Provided are imaging methods for detecting programmed death ligand 1 (PD-L1): (a) A peptide with binding specificity for PD-L1 and a reporting domain An imaging agent comprising a conjugate and, optionally, a linker, as described immediately above. The reporter molecule, if present, links the peptide to the reporting moiety, and the linker, if present, 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 of the fibronectins; (c) contacting the cell or tissue with an imaging agent; and (b) generating an image to detect PD-L1. Process.

[0048] As used herein, the term "imaging" or "creating an image" refers to Visualize detectable compounds by measuring the energy emitted by the compound In some embodiments, the term "imaging" refers to the use of any imaging technique. The term is used to measure 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 refers to the use of detection from outside the subject. In some embodiments, the image includes administering to the subject a compound capable of detecting the It is produced by differences in the spatial distribution of the contrast agent that accumulates in different locations in the subject. In an embodiment, administration of the contrast agent is by injection.

[0049] The term "imaging agent" refers to a substance that can be used to produce images, for example by positron emission tomography (PET). As used herein, "positron emitting" is intended to include compounds that are capable of "Positron Emission Tomography Imaging" or "PET" stands for "positron emission tomography imaging." systems or equivalent, and any device 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. The present invention can be implemented using variations of the above 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 site, the imaging agents of the present disclosure can be used in a variety of imaging applications, including PET, single photon emission computed tomography (SEM), and other imaging modalities. In tomography (SPECT), near infrared (fluorescence), photoacoustic, and Raman imaging It can be used.

[0051] In some embodiments, imaging involves detecting the presence or absence of a subject or The entire patient or specific areas of the subject or patient are scanned to detect the signal. The detected signal is then converted into an image. The resulting image can be used by, for example, a physician. The images should be interpreted by a trained observer. Generally, the images are taken about 1 minute to about 20 minutes 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 being used. In certain embodiments, imaging is performed between about 1 minute and about 4 hours after administration, for example, 15 minutes. Between 30 minutes and 45 minutes, Between 45 minutes and 60 minutes, Between 60 minutes and 90 minutes In some embodiments, detection of PD-L1 is performed between 60 and 120 minutes. is performed no sooner than about 60 minutes after administration of 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 imaging of peptides labeled with I-124. This may be done 24 hours after injection.

[0052] Once an image is obtained, one of skill 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 may be treated. The extent of the condition or the effectiveness of a treatment the subject is receiving can be determined.

[0053] In some embodiments, contacting the cell or tissue with the imaging agent is performed in vitro, i.e. "Contacting" refers to any of the above-mentioned methods of the present disclosure. 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 includes placing the imaging agent and cells or by introducing and preferably mixing tissues in vitro or ex vivo In some embodiments, the method is performed in vivo. In one embodiment, the contacting comprises 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 present invention refers to administering 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 the techniques described herein, e.g., positron emission tomography (PET). required 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 particular This can vary depending on factors such as the compound. For example, the choice of compound can determine what constitutes an "effective amount." Those of ordinary skill in the art would be able to determine the extent to which the present invention can be applied without undue experimentation, considering the factors contained herein. A determination as to the effective amount of the compound can be made without the need for further study.

[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 include those encompassed by the term "subject." It should be understood that the subject invention is effective with respect to all vertebrate species that are intended to be included therein. Thus, a "subject" is a person who is undergoing medical treatment, 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, and macaques. Mammals, including but not limited to: cattle, such as cows, cows, etc.; sheep, such as sheep, etc.; goats , for example goats; swine, for example pigs and hogs; equines, for example horses, donkeys, zebras Cats, including wild and domestic cats; Canines, including dogs; Lagomorphs, rabbits, 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. The present invention 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, etc. In some embodiments, the subject is a human, a rat, a mouse, a cat, a dog, a cow, a cat ... A horse, sheep, cow, monkey, bird, or amphibian.

[0056] In general, the imaging agents of the present disclosure can be administered to treat a disease, disorder, or condition by any suitable route of administration, including: or may be administered to a subject for detection of a condition: orally, nasally, mucosally, ocularly, rectally, Intravenous, intramuscular, subcutaneous, or intramedullary injection, including vaginal or parenteral, as well as intrathecal, 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. Intravenously and sublingually, transdermally, via inhalation spray, or other methods known in the art. Other modes of delivery.

[0057] As used herein, the terms "systemic administration," "systemic administration," "peripheral administration," and "peripheral administration" are used interchangeably. The term "administered" refers to the fact that they enter the subject's or patient's system and thus undergo metabolism and other similar processes. This refers to administration of the composition to undergo 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, intravesical, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, Including, but not limited to, subcapsular, 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 the 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 may grow locally within an animal. They may be present locally or circulate in the bloodstream as separate 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, tracheal cancer, and bronchial cancer. Ripple-negative breast cancer and other breast cancers, prostate cancer, colon cancer, esophageal cancer, stomach cancer, white blood cell cancer, 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. be.

[0061] As used herein, a "tumor" refers to any neoplastic cell, whether malignant or benign. This refers to the growth and proliferation of cells and tissues, as well as all precancerous and cancerous cells and tissues. As used in the specification, 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 of any kind, can be detected using the subject matter of the present disclosure. As used herein, the term "infection" refers to an organism that causes a disease. the invasion of the body tissues of host organisms by fungi, their proliferation, and the development of pathogens and their products. It refers to the reaction of host tissue to the toxins produced by the bacteria. Infections include hospital-acquired infections, surgical infections, and 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 following the use of immunosuppressants, cancer chemotherapy, radiation, 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 infectious disease is disseminated tuberculosis. In some embodiments, the infectious disease 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 inflammatory bowel disease include asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, and bronchitis. Utriculitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis media, pelvic Intracellular inflammatory disease, 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 the

[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 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 treatment-induced changes in PD-L1 expression. Such methods detect and / or measure changes in PD-L1 expression, such as PD-L1 expression in a specific To determine the efficacy of a treatment method and / or to determine an effective therapeutic dosage range. can be used for.

[0066] III. Kits Containing Contrast Agents In some embodiments, as described above, the subject matter of the present disclosure relates to a method for treating apoptosis with programmed death ligand 1 (P 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, the linker, when present, being a peptide and the reporting moiety, and in the absence of a linker, includes an imaging agent that is 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 method includes instructions on how to carry out the method. 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 any 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, the 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 include other substances useful for

[0069] The container may contain a composition of the present disclosure or other material useful for carrying out 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 of a material such as a stopper, a stopper and The container is sealed with a crimp seal or a 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 related to the subject matter of this disclosure. The selection can be made by one of skill in the art without undue experimentation.

[0070] In an embodiment, 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. and instructions, and / or other information regarding use of the composition. The box is constructed from sturdy materials such as cardboard and plastic and includes instructions and other information. The kit may include multiple containers containing the compositions of the invention. In such a kit, each container is the same size as each other container and contains the same amount of the composition. Alternatively, different containers may be of different sizes and / or contain different amounts of The container size and contents may include a composition or a composition having different ingredients. Numerous different configurations of the present invention are contemplated by the present invention, and therefore all permutations are included herein. As will be readily appreciated, the various aspects of the invention do not necessarily need to be specifically listed in the book.

[0071] Although specific terms are employed herein, they are used in a generic and descriptive sense only. 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 those of 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 convention, the terms "a," "an," and "the" are used to denote portions of a patent claim. When used in this application including ranges, it refers to "one or more." Thus, for example, For example, "subject" should be used unless the context clearly indicates the opposite (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 grammatical variations thereof are used to indicate that the enumeration of items in a list is consistent with the enumerated The terms are non-exhaustive 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, proportions, shapes, compositions, parameters, percentages, parameters, quantities, characteristics, arrangements, Other numerical values ​​used in this specification and claims are expressly defined as being within the meaning of the term "about" In all cases, even if not expressly appearing in conjunction with a quantity or range, the term "about" is used. Therefore, unless indicated to the contrary, the following disclosure The numerical parameters set forth in the specification and attached claims are not precise and should not be construed as limiting the present invention. It does not have to be, but may 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 of skill in the art that depend on the desired properties being achieved. 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 ±100%, in some embodiments ±20%, 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 carrying out the disclosed methods or employing the disclosed compositions. "Inclusion" can mean inclusion where appropriate.

[0075] Additionally, the term "about" may be used in conjunction 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 the Modify the range by extending the boundaries above and below the specified numbers. A recitation of the formula "a" includes all numbers subsumed within the range, e.g., all fractions thereof subsumed within the range. The whole number range (for example, the enumeration 1 to 5 includes 1, 2, 3, 4, and 5, as well as their decimals) , e.g., 1.5, 2.25, 3.75, 4.1, etc.) and any range therein. Contains a range of. EXAMPLES

[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 intended to be limiting in any way. Thus, this should not be construed as limiting in any manner in which the disclosed compounds are prepared. isn't it.

[0077] Example 1 (Rapid tumor PD-L1 detection with PET using highly specific peptides) 1.1 Background: Increased PD-L1 expression in the tumor microenvironment (TME) mediates active immune infiltrates Immunity via binding to the programmed cell death protein 1 (PD-1) receptor expressed by It causes immune suppression by inactivating immune infiltrates (Okazaki et al., 2007, and Topalian et al., 2015). PD-L1 expression on tumor cells and in the TME is associated with increased risk of developing PD-L1-associated 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 for use in the US Food and Drug Administration (FDA). Recently approved by the Food and Drug Administration, PD-L1 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. is the most studied predictive biomarker for PD The available FDA-approved diagnostic IHC tests for -L1 have significant limitations and ch et al., 2016; Mansfield and Dong, 2016; and Phillips et al., 2016. Ips et al., 2015, inconsistencies in antigen positivity definitions, discrepancies in detection antibodies, and lack of agreement between assays adequacy, as well as intra- and inter-tumor heterogeneity that compromises accuracy and reliability, and therefore treatment In addition, tissue samples obtained by biopsy for testing are often not available. 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 resistance 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 available within 60 minutes of administration. Novel PET contrast agents that perform well within the standard clinical workflow of imaging in PD -overcome the shortcomings of available (IHC-based) methods for assessing L1 expression status Wax.

[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. The serotypes are promising for clinical application due to their fast clearance and ease of synthesis. Somatostatin is a desirable candidate (Reubi et al., 2008; Sun et al., 2016). Peptide-based PET targeting receptor and chemokine receptor 4 (CXCR4) The racer produces a high target-to-nontarget 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 6039749, Macrocyclic Inhibitors of the PD -1 / PD-L1 and CD80 (B7-1) / PD-L1 Protein / Pro tein Interactions;Published on June 23, 2016, Mapel Li et al., International PCT Patent Application Publication No. WO2016 / 100285, Immunomod Published on June 23, 2016, the International PCT Patent Application of Sun et al. 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 remains unclear. These PD-L1 binding peptides rapidly increase PD-L1 expression in tumors. It was hypothesized that it would be possible to detect HIV-1 infection with high specificity and accuracy. The reported peptide library has a single primary amine that is most suitable for jugation. 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, conjugates were attached to WL12 and 64 Cu WL12 (Eisenwiener et al., 2000) and expressed a peptide against PD-L1. We evaluated the binding affinity of the tide derivatives and compared their binding affinity to PD-L1 in cell lines with variable PD-L1 expression. 64 Cu ]WL12 in vitro uptake was evaluated. As a proof of concept, [ 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) ) tumors and syngeneic negative control tumor (CHO)-bearing NSG mice. [ 64 The tissue distribution and target specificity of [Cu]WL12 were examined by 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 that of PD-1. To evaluate the binding mode to D-L1, we docked WL12 instead of PD-1. To investigate the mechanism of action of PD-L1 in the regulation of PD-1, we have developed a co-crystal structure of human PD-L1 bound to PD-1 (PDB ID: 4ZQK) (Za Considering the structural complexity of the macrocyclic molecule WL12, the present inventors We first performed a conformational search and used Glide to identify the PD-1 binding site on PD-L1. The 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 1C). 2) When the structure of PD-1 was superimposed with bound WL12, the binding between the two was clearly evident. The two base domains of PD-1 that form the binding interface with PD-L1 are similar in mode of binding. The Il chain of WL12 overlaps with the pseudochain of WL12 (Fig. 1C). The L-leucine of WL12 binds to the Il chain of PD-1. It is inserted into the same small hydrophobic pocket as e134 and contains 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 glycine amide forms a hydrogen bond with the backbone of Gly120. The serine hydroxyl interacts with Gln66. The ornithine residue is exposed. Without wishing to be bound to 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-dependent IL-1 binding inhibitor does not interfere with WL12 binding to PD-L1.

[0082] 1.2.2:[ 64Cu]WL12 inhibits PD-L1-specific cell uptake in vitro Indicates inclusion. 13 Conjugating DOTAGA using the ornithine (Orn) primary amine This is then used to gate the non-radioactive C 2+ Prepare an analogue (WL12-Cu) 6 4 The resulting WL12D and the corresponding WL12-Cu were radiolabeled with Cu. and characterized by mass spectrometry (Figures 2, 3, 4, 5, 6, and 7). 7) and were subjected to in vitro evaluation. WL, which inhibits the interaction of PD-L1 with PD-1, The half-maximal inhibitory concentration (IC 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 23, respectively. 23, and an IC of 2.9 nM 50 The values ​​were observed (FIGS. 8A, 9, 10 and the following). Table 1). These data are 13 Orn side chain was modified with DOTAGA and Cu 2+ Replying to @sarah_mcdonald These results indicate that WL12 retains high binding affinity to PD-L1 even after transformation. There are.

[0083] [Table 1]

[0084] To demonstrate PD-L1 specificity and cellular uptake, 64 Cu]WL12 high Produced with specific activity (1.9 ± 0.1 mCi / μg) and radiochemical purity (>95%) (Figures 11 and 12). 64Cu]WL12 incubated with hPD-L for 1 hour 1 cells showed >50% uptake of the incubated dose compared to negative control CHO cells. hPD-L1 cells were then cultured in a 3x100-well platelet-free control, demonstrating a 43-fold increase in both detected and bound radioactivity (Figure 8C). 64 Cu]WL12 alone or in the presence of a blocking dose of 1 μM WL12. The binding specificity was tested by incubating the antibody with 64 Cu]WL12> A 95% reduction in the expression of PD-L1 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 for the tumor (Figure 8C). 64 Cu ]WL12’s performance was evaluated in two triple negative tumors with high and low PD-L1 expression, respectively. and in the transmembrane biliary (TNBC) cell lines, MDAMB231 and SUM149. (Figure 8B). Radioactivity in MDAMB231 cells compared to SUM149 cells. The two-fold higher uptake of PD-L1 was 64 Further confirming the specificity of Cu]WL12 Flow cytometry analysis of PD-L1 expression showed that the mean 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). These results are 64 Cu]WL12 is dependent on PD-L1 expression in vitro They have demonstrated that it binds to cancer cells.

[0085] 1.2.3:[ 64 Cu]WL12 specifically accumulates in tumors with high PD-L1 expression. [ 64To 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. The PET imaging study demonstrated that hPD-L1 tumors 64 Cu]WL The increased uptake in hPD-L1 tumors was seen 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. 64 Cu] 1 and 2 h after WL12 injection (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 were immunized at 14.9 ± 0.8 injected dose / g (%I 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 relatively high, and the concentration of 100 mg / g was 4.0±0.6% ID / g (Figure 17). 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 26.6 ± 1.2, respectively. and 4.7±1.2, [ 64 Cu]WL12 shows high signal for PD-L1 specific image This was consistent with the ability to provide a good noise ratio (Figures 15A and 15B).

[0086] Biodistribution studies performed after 2 hours showed reduction of radioactivity in the kidneys, liver and tumors. The results showed similar profiles with a trend toward a 100% specific antibody titer (Figure 17). In order to 64 Cu]WL12 was administered simultaneously with excess WL12 (50 μg, 2 mg / kg). The %ID / g values ​​of hPD-L1 in tumors were > A 75% reduction (P<0.0001) was observed, with no significant difference observed in control CHO tumors. There was also a decrease in the amount of radioactivity taken up by the kidneys. There were no significant differences in the uptake of radioactivity in other tissues. No significant 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 improve renal clearance of peptides. These tumors are known to express PD-L1 and are associated with increased 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, mouse P These results suggest that the antibody has very low or no affinity for D-L1. [ 64 Cu] As further support for 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 carried out jointly by [ 64Cu]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 molecular weight of the chromatogram, CD spectroscopy was performed in combination with water, DPC, and SDS. Thus, Trp residues are present in the CD spectrum of the WL12 peptide in the region of 220–240 nm. In surfactant-free solutions, the minimum is at about 220 nm and the A positive shoulder was observed at 30 nm. Addition of surfactant reduced both bands. The bands are slightly red-shifted and the latter shows an increase in intensity. Both Trp chromophores are in close proximity and form a single adsorption As a result, their excited states interact with each other, 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 is the in-phase combination of the two monomer excitations, and the other arises from heterophase bonding (Grishina 1994, and Kelly 2000).

[0088] CD spectra of disordered peptides are 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 was observed for 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, Trp staining in the far-UV CD spectrum of WL12 The strong contribution of the nucleotide sequence to the secondary structure may affect the accuracy of the 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 by PET in vivo and in vivo. 64 Cu]WL12 pharmacokinetics and Biodistribution was assessed by imaging patients with PD-L1 detection within 60 minutes of radiotracer administration. This suggests that it is feasible to fit the device into the standard clinical workflow for Rapid, non-invasive detection of PD-L1 expression across all malignancies may be an important tool to assess immune regulation. It offers an unprecedented opportunity to stratify patients for therapy.

[0090] 1.4 Materials and Methods 1.4.1 Materials: The PD-L1 binding peptide, WL12, was purified using CPC Sc 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-Dimethylamino) xotetrahydro- 2H-pyran-3-yl)-1,4,7,10-tetraazacyclododeca Dot-1,4,7-triyl)triacetic acid (DOTAGA anhydride) and64 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 Catalog Co., Ltd. (blocking number 1086-PD-050) and recombinant human PD-L1 (B7-H1)-His-tag The protein (catalog number 9049-B7) was purchased from R&D systems (Minneapolis, MN). , Minnesota).

[0091] 1.4.2 Docking study: To perform docking of WL12 to PD-L1, The crystal structure of human PD-1 bound to PD-L1 (PDB ID: 4ZQK) was used as a template. The model was Protein Preparation in Maestro. Wizard in Maestro (Schrodinger Release 2 016-2: Maestro, version 10.6, Schrodinge r, LLC, New York, NY, 2016) was used to prepare the Astry et al., 2013). This involves the assignment of bond orders and formal charges, the attachment of hydrogen atoms, The hydrogen bond network within the protein is optimized by adding the missing side chains. (reorientation of thiol and hydroxyl groups, sanitization of Asn, Gln and His side chains) including molecular dynamics prediction, as well as prediction of the protonation states of His, Asp, and Glu), followed by A simple minimization is performed using the Prime Conform ational Search (Schrodinger Release 2016 -2: Prime, version 4.4, Schrodinger, LLC , New York, NY, 2016) to determine the conformational structure of WL12. A locus 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 aqueous solution without surfactant and in dodecyl In aqueous micellar solution of phosphatidylcholine (DPC) and sodium dodecyl sulfate (SDS) The CD spectra of the peptides in mixed DPC:SDS micelles with a molar ratio of 5:1 were 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 ) versus wavelength λ (nm). The content of secondary structure was determined by CO 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 1 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 measured using a RP-HPLC system (Varian ProStar). Lent Technology 1260 Infinity Photodiode Array Detector (Agilent Technologies, Wilmington, DE) A semi-preparative C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Toray) was used. (France, CA) and 98% H2O (0.1% TFA) with 2% MeOH ( Starting with 0.1% TFA) and reaching 100% MeOH in 60 min at a flow rate of 4 mL / min Gradient elution was used to purify the desired WL12D, which was collected at 44.5 min and evaporated. Dissolve in deionized water and freeze-dry 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 using a fluorochemical microscope (H. Ltronics, Billerica, Massachusetts) (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, pH = 4.5 adjusted with glacial acetic acid). The mixture was dissolved and 55 μL of 0.02 M CuCl2 aqueous solution (1.1 μmol) was added. The combined reaction mixture was incubated at 65°C for 30 min and incubated for 30 min 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, PD-L1 and P WL12-Cu was used as a standard for the D-1 competitive binding assay. 2+ RP-HPLC conditions using the complex The conditions were optimized (Figure 7). Theoretical 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: Competitive inhibition assays were performed in discussion with Cisbio (Woodard et al., 2014). , optimized from a previously described fluorescence resonance energy transfer (FRET)-based assay. All Binding / inhibition assays were performed in 21 µL of FRET assay buffer (dPBS, bovine serum albumin). (0.1%, w / v), Tween-20 (0.05% v / v) and sodium fluoride The assay conditions were initially set at PD-1 and PD-L1 concentrations PD-1-Ig was optimized at final concentrations of 10 nM, 20 nM and 40 nM. with PD-L1-His-tag at final concentrations ranging from 0.65 to 320 nM (each concentration in triplicate). for 15 min, followed by anti-human IgG-Eu 3+ 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 h of incubation, 1 μL of NaF assay buffer was added (final concentration, 40 0 mM), and the plate was inserted into 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 addition of PD-1-Ig (final concentration 20 nM) was added in 5 μL of assay buffer and incubated for 15 min. Then, IgG-Eu (final concentration 2 nM) and anti-6HIS-XL665 (final concentration 40 nM) were After incubation at room temperature for 1 hour, 1 μL of assay buffer containing 0.5% MgCl was added. 1 L of NaF was added (final concentration 400 mM) and the plate was incubated with a Perkin Elmer V The data were read on an 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 preparation: purchased from University of Wisconsin 64 Cu By evaporating Cl2 to a small volume and titrating with 0.1M sodium acetate solution 64 C u(OAc) 2 For radiolabeling, approximately 1:1 in 100 μL of sodium acetate was 1.0 μg of WL12D peptide conjugate (4.27 nmol) was added to approximately 185 MBq ( Approximately 5mCi) 64 Cu(OAc) 2 and incubated at 65°C for 30 minutes. The resulting radioactive tracer was analyzed using C-18 (Luna, 5 μm, 10 x 250 mm; Ph enomenex) semi-preparative column, equipped with a radioactive single channel radiation detector. arian ProStar system (Model 105S; Bioscan, Poway, CA) and a Varian ProStar UV absorbance set at 280 nm The purification was performed using a 98% H detector. 2 O (0.1% TFA) and 2% MeOH (0.1 % 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 rate of 1.9 ± 0.11 mCi / μg with a yield of 52.09 ± 6.3%. Obtained by 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 were purchased from the Canned 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. The gene was provided by the Johns Hopkins Genetic Resource Facility and verified by STR profiling. SUM149 cells were cultured in 5% FBS, 1% P / S, and 5 μg / mL insulin. and 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 purpose was 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 harvested by centrifugation and adherent cells are isolated. Cells were dissociated in enzyme-free PBS-based cell dissociation buffer (Thermo Fisher Scientific). Cells were detached using a centrifuge (Ecientific, Waltham, MA). Add 1x PBS containing 2 mM EDTA and 0.5% FBS in 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 anti-human PD-L1 1 antibody (BD-MIH-PE, clone number MIH1, catalog number 557924, Bec ton Dickinson, Franklin Lakes, New Jersey) Flow cytometer stained according to manufacturer's protocol and analyzed on a FACSCalibur The samples were analyzed using a Becton Dickinson chromatograph. At least 20,000 The event was recorded.

[0100] 1.4.9. In vitro binding: hPD-L1, CHO, MDAMB231 and [ 64 Cu]WL12 in vitro binding was measured using 1 μCi of radiation 1x10 Sex Tracer 6 Determined by incubating with cells for 1 h at 37°C. After incubation, the plate was counted using an automatic gamma counter (1282 Compugmam a CS, Pharmacia / LKB Nuclear, Gaithersburg Maryland Cells were washed three times with cold PBS before being counted at 4°C (Denver). 64 Cu]WL12P To demonstrate D-L1 specific binding, 1 μM of WL12 peptide or humanized anti-PD-L PD-L1 blocking was performed with the antibody atezolizumab. The mean fluorescence intensity values ​​were calculated by incubation. All cellular uptake studies were performed in a 3-mL 10-mL PBS-MS ... Each cell line was performed in triplicate and repeated three times.

[0101] 1.4.10. Animal Models: Animal studies were performed at the JHU Animal Care and The study was conducted according to a protocol approved by the ACUC. Six to eight week-old female non-obese diabetic severe combined immunodeficiency gamma (NSG) mice were cultured at JHU Obtained from the Immune Compromozed American Core. 10×10 on both sides of the upper abdomen 6 CHO-PDL1 and CHO cells were implanted subcutaneously. Tumor is 200-300 mm 3 When the volume reaches 100 μm, the mouse is imaged or in vivo It was used in the intracellular distribution experiment.

[0102] PET-CT imaging of mouse xenografts: Mice were dosed 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. Fluorane levels were maintained at 100 nm. 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. A 512-projection scan was performed. The PET data were analyzed using a two-dimensional regular subset-expectation maximization (EM) algorithm. The reconstruction was performed using the algorithm (2D-OSEM), and the dead time and radioactive decay were 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 accomplished using a 350 nm NMR spectrometer (Hillsboro, OR).

[0103] 1.4.12.Ex vivo biodistribution: high and low PD-L1 expression ( n=5) were administered 40μCi 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 counted in an automated gamma counter (Per Elmer-2480 Automatic Gamma Counter - Wizard2 3´´Walla For blocking tests, mice were given 2 mg / kg (50 μg) of untreated The peptide was co-injected with the radioactive tracer. Percentage of injected dose per gram of tissue Percentage (%ID / g) values ​​were calculated using signal decay correction and external [ 64 Cu] standard The biodistribution data presented are the mean ± standard deviation of the mean. Error bars are mean squared mean (SEM).

[0104] 1.4.13. Data Analysis: Prism 6 software (GraphPad software) Statistics were analyzed using unpaired two-tailed t-tests using the National Institute of Standards and Technology, 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 50 Oh and Ki values ​​were calculated using Prism 6 software (GraphPad).

[0105] Example 2 (PD-L1-directed PET to develop PD-L1-targeted drugs) 2.1 Overview: Cancer immunotherapy (CIT) has demonstrated durable responses in a variety of malignancies. However, immune checkpoint targeting therapy has been shown to improve patient survival rates. Nearly 70% of patients treated with this 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 for combination checkpoint therapy to improve survival, but In many cases, increased knowledge of combination strategies comes at the expense of increased immune-related adverse events (irAEs). These results suggest that addition of riboflavin 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. Thus, one aspect of the subject matter of the present disclosure is the development and Develop strategies for using PD-L1-based PET imaging in the treatment 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 therapeutic drugs (antibodies, peptides, small molecules) in relevant in vivo tumor models and establish using PD-L1 PET imaging.

[0106] 2.1.1. Advances enabled by PET-based quantification of PD-L1 dynamics: NSC Targeted therapeutic AtzMab for PD-L1 in LC, TNBC and colon tumors and its mouse counterpart It has recently been discovered that accumulation of mela (PRO) is not entirely dependent on PD-L1 expression. However, H2444 NSCLC xenografts with high PD-L1 expression were identified by IHC and Breast cancer xenografts with low PD-L1 expression as detected by flow cytometry Accumulated significantly less radiolabeled AtzMab relative to that seen in the explants. (Chatterjee et al., 2016). Similarly, in a syngeneic mouse tumor model In the present study, radiolabeled PRO injected systemically was primarily associated with tumor vasculature and was expressed in 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 agents within the tumor, This is an important factor in treatment resistance (Goel et al., 2011). In order to achieve this, PD-L1-targeting drugs that act on tumor cells and tumor immune infiltrates can be made to approach tumor cells. Therefore, WL12 / [ 18 F]WL12 and other peptides Due to their very small molecular size, radiolabeled peptides, such as ribozymes, are difficult to label with antibodies. It is therefore possible for the medicament to penetrate tumor tissue and reach target cells more effectively and efficiently than the medicament. By using the analysis and correction, 18 F]WL12 measurement or similar radioactivity Measurements performed using labeled peptides showed that the desired occupancy rate in tumor tissue was observed in target tumor cells. This may aid in identifying / optimizing the therapeutic mAb dose required to achieve

[0107] Therefore, PD-L1-directed PET has been applied to PD-L1-targeted drug development. In order to assess the potential value, [ 64 Cu]WL12 was used. Therapeutic PD-L1 antibody atezoside in relation to dose versus mAb localization in tumors as seen by PET To evaluate and compare 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 In addition, such PD-L1 PET measurements could be used to detect PD-L1 expression at the tumor site. By enabling quantification of their potential target engagement in This may guide the future development of novel PD-L1-targeting therapeutics.

[0108] 2.1.2. Innovations in the use of PD-L1 PET in drug development and evaluation: The presented innovative PD-L1 peptide-based PET imaging strategy is a promising candidate for current and future Target engagement efficacy of anti-PD-L1 therapeutics in the tumors where they are most relevant Allows assessment of potency (i.e. occupancy and residence time). Dynamic PD-L1 density / turnover The extent of PD-L1-expressing tumor burden, which influences serum mAb concentrations, may be related to complete tumor perfusion. Together with the overall efficacy and resulting intratumoral mAb accumulation, this significantly impacts therapeutic efficacy. Radiolabeled antibodies were used to define required mAb dose levels and to calculate target surface molecule occupancy. Although it has been previously used to study the effects of schizophrenia on cellular function (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. Our method effectively addresses this issue by quantifying PD-L1 occupancy at the tumor site. The relationship between effective mAb dose and cumulative dose achieved was evaluated using key tumor physiology parameters. In addition to considering the contribution of Measurement of PD-L1 expression 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 guidance on strategy.

[0109] (2.1.3. Usefulness of PD-L1-PET in the development and evaluation of PD-L1-targeted drugs Rate the sex :) (2.1.3.1 Rationale) Therapeutic antibodies targeting PD-L1 and PD-1 are being used to treat patients with PD-L1-positive tumors. At the doses currently used, the drug 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 in tumors is dynamic. However, this is still poorly understood (Brahmer et al., 2012). Studies have found that in some tumors, PD-L1 antibodies are 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 support 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 help improve This suggests that informed PD-L1-directed therapy is needed. Without wishing to be bound by any of the above, PD-L1 PET is a method to detect PD-L1 Such PK measurements of peptides and small molecules are evaluated with respect to target binding and residence time. In addition, PET-based dosing is considered to be a valuable tool for -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 tumor function. It is thought that this will lead to

[0110] 2.1.3.2 Representative Data: Radioactivity of Available Anti-PD-L1 Antibodies and PD-1 Derivatives The labeled version has been used to non-invasively detect PD-L1 expression (Cha tterjee et al., 2016; Deng et al., 2016; Hettich et al., 2016; osefsson et al., 2016; Lesniak et al., 2016; Heskamp et al., 20 15; Maute et al., 2015). To this end, we are developing a therapeutic antibody, AtzMab, that The specificity of its PD-L1 detection was examined in immunocompromised and immunosuppressed mice. Human TNBC and NSCLC xenografts in mice and the 4T1 syngeneic breast tumor model In slices, PET, SPECT and optical imaging were used to demonstrate (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 anti-inflammatory drug that is effective in treating progressive or Metastatic bladder cancer, (Powles et al., 2014) melanoma, (Hamid et al., 2013) NSCLC, (Spigel et al., 2013) RCC, (Cho et al., 2013) TNBC, It is under clinical evaluation for the treatment of cancer of the lungs and several other cancers.

[0111] Accumulation of radiolabeled AtzMab in tumors was consistent across both cancer types (NSCLC and TN BC) was found to be PD-L1 specific (Chatterjee et al., 2013). 016; Lesniak et al., 2016). 111I n]AtzMab also accumulates in tumors , and was found to be not entirely dependent on PD-L1 expression, which was due to factors such as interstitial fluid pressure, tumor convection, suggest that spatial variation in vascular extravasation and extravasation are some of the contributing factors. This is a problem often observed with antibodies (Baxter et al., 1989). 31 TNBC xenografts outperform 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 immunohistochemistry 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 multiple mechanisms that influence antibody distribution in tumors. 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 evaluating 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 unresectable AtzMab therapies can be evaluated at tumor sites where they are needed. This provides an unexpected tool for understanding the distribution of PD-L1 antibodies in tumors. These improvements may have implications for clinical antibody dosing and therapeutic monitoring. Therefore, we evaluated the binding of AtzMab to PD-L1 in tumors. 64 Cu]WL12-P The ET's capabilities were tested. 64 Cu]WL12-PET and biodistribution studies As quantified, accumulation of radioactivity in hPD-L1 tumors was significantly greater with AtzMab (20 mg 21A, 21B, and 21C. 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 for human PD-L1 (Lesniak In vitro binding studies demonstrated that unlabeled WL12 binds to C Concentration-dependent inhibition of y5-conjugated AtzMab binding to PD-L1 and 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. Taken together, these results confirm that the binding of WL12 and AtzMab is Demonstrating overlapping sites, AtzMab targeting in PD-L1-expressing tumors To assess engagement and dwell time (target engagement efficacy) 64 This approach is applicable to PD-L12-PET. The results also extended to cancer cell lines with naturally elevated IL-1 expression. 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 contrast agent uptake in mice receiving the treatment was also observed.

[0113] The application is similar to other PD-L1-targeting therapeutic antibodies such as Avelumab (AvMab). AvMab is being investigated in the treatment of NSCLC (NCT02395172), advanced RCC and A human IgG1 antibody currently undergoing multiple Phase III clinical trials in several cancers, including gastric cancer. Analysis of the crystal structure of PD-L1 in complex with AvMab revealed that A vMab targets several 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 immunization 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 methods for 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 used to treat Nearly one in four clinical trials involving checkpoint inhibitors have been included. levels, occupancy by their PD-L1 therapeutics, and ensuring optimal immune responses The relationship between the extent and duration of target engagement in tumors and dosing The occupancy rate of PD-L1 in tumors is a function of the dynamic changes in PD-L1 expression, if any. and by tumor intrinsic and 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 tumor necrosis factor-1 (PD-L1) expression, Radiolabeled PD-L1 binding peptides were investigated. Structural analysis was performed on peptides and therapeutic monoclonal antibodies. and overlap in the interactions of mAbs with PD-L1, and the therapeutic potential of tumors This made it possible to measure the occupancy rate of mAb using positron emission tomography (PET). PET imaging and biodistribution studies in multiple xenograft models have demonstrated variable We show that PD-L1 expression in patients with PD-L1-positive leukemia and its saturation by a therapeutic PD-L1 antibody can be quantified. In addition, 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 P 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 optimizing

[0115] More specifically, the subject matter of the present disclosure relates to quantitative positron emission tomography (PET) imaging. Using ELISA, we assessed PD-L1 expression levels and PD-L1 expression in tumors in vivo. Addressing the need to characterize mAb target engagement in tumors Repetitive measurements of target expression in a wide range of conditions (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 the method has not been achieved (Peterson et al., 2008; Linden et al., 2009). 06).

[0116] It binds to human PD-L1 with high affinity and specificity and is 1 s Generates high-contrast images in under 20 minutes 64 Small peptides radiolabeled with Cu , [ 64 Cu]WL12 has been recently developed (Chatterjee et al., 2017). The embodiment is a method for detecting PD-L1. 64 Cu]WL12-PET was described and used to evaluate the efficacy and safety of WL12 in lung cancer and To quantify the dynamic changes of PD-L1 expression in experimental models of breast cancer. A-approved mAbs, atezolizumab, avelumab and durvalumab (DurMab) Evaluate PD-L1 engagement in patients with 64 The ability of Cu]WL12 PET was evaluated. In addition, the effect of PD-L1 engagement on the extent and duration of tumor PD-L1 engagement 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 (PDL). 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 that inhibit PD-L1:PD-1 interactions Clonal antibody therapy (mAb) is currently in clinical trials, and patients receiving these treatments Nearly 30% have a sustained response (Topalian et al., 2015; Lipson et al., 2016). 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 action of anti-PD-L1 mAbs is 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 results using peripheral blood mononuclear cells (PBMCs). The PD-L1 antibody BMS-936559 was administered at doses ranging from 0.1 to 10 mg / kg. Uniform target occupancy of 64–70% has been reported for each dose (Brahmer et al., 2013). The location of PD-L1 mAb at the most relevant site, i.e., the tumor, and and the degree and duration of target engagement to ensure optimal immune responses. Much remains unknown about the relevance of intergroup administration.

[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 characterize the immune-tumor microenvironment (TME) and intra- and inter-tumor heterogeneity of PD-L1 expression However, this has serious limitations as it cannot 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 therapeutics, 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 0:20 nM) (Chatterjee et al., 20 17) Initial molecular modeling analysis has identified important molecular interactions, including PD-L1:W L12 and PD-L1: 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) 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 to 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 block 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 Fig. 34A), the overlapping region (cyan) is a deep pocket. and acts as an anchor point for all interaction points. zMab (red) is a marker for 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 ester of the antibody. PD-L1 was then constitutively expressed in CHO cells (Cho- hPD-L1) and MDAMB231 breast cancer cells that naturally express PD-L1 (Chat Terjee et al., 2016) performed a competitive inhibition assay with WL12. WL12 dose-dependent inhibition of Cy5-PD-L1 mAb binding to PD-L1 at inhibitory concentrations of 100 μM The toxicity was 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. Incubated with fluorescent versions of AtzMab, AveMab and DurMab. Flow cytometry showed a significant decrease in binding fluorescence (P<0.001) with antibody-PD-L The ability of WL12 to disrupt CX3+ / CX4+ / CX5+ / CX6+ / CX7+ / CX8+ / CX9+ / CX1 interactions was further demonstrated (Figures 34C and 34D). No change in binding fluorescence was observed when using the CR4-specific antibody MDX1338 Further confirmation of the specificity of the WL12:PD-L1 interaction was obtained. (HCC827, H226, MDAMB231 and hPD-L1) and PD-L1 Negative (Sum149 and CHO) cells were treated with WL12 analogs radiolabeled with 64Cu ( [ 64 The WL12 analogs inhibited hPD-L1 / High affinity (IC50<2 in vitro and in vivo in CHO cells) It has previously been demonstrated to bind PD-L1 with potency (0 nM) and selectivity, but has 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 an additional check on efficacy, 60 nM mAbs were used compared to PBS-treated controls. When treated with PD-L1, the [ 64 Cu]WL12 uptake significance A 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 by PD-L1 mAb. 1 This shows that engagement can be monitored.

[0122] 3.3.2 Quantification of tumor PD-L1 engagement by AtzMab. To non-invasively assess PD-L1 engagement by therapeutic mAbs in vivo To investigate the efficacy of IFN-γ in NSCLC xenograft models, we tested these models. Approximately 50% of patients were PD-L1 positive, and PD-L1 IHC was negative for immune checkpoint therapy. It was selected because it is used as a predictive biomarker for NSCLC patients with Mansfield et al., 2016). H, which shows low and intermediate PD-L1 expression, respectively. NOD scid gamma mice bearing 226 and HCC827 cell-derived xenografts ( Figure 36A) shows a single dose of AtzMab administered intravenously (20 mg / kg, 24 h) They were dealt with in . 64 PET images acquired 2 hours after [Cu]WL12 injection show 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 control subjects (Figure 35A and (Figure 35B). The 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 saline The [ 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. (Figure 35C and Figure 35F). The results are 64 Cu]12 by AtzMab To demonstrate that it is possible to quantify in vivo targeting of PD-L1 in tumors .

[0123] Efficacy of a single dose of AtzMab to target different PD-L1 levels in tumors To evaluate efficacy, we used C-cell lines with 4-10 times higher PD-L1 expression than NSCLC cells. PET and biodistribution studies were performed in tumors derived from the HO-hPDL1 cell line (Figure 1). 36) CHO-hPDL1 / C cells treated with AtzMab (20 mg / kg, 24 hours) HO tumor-bearing mice showed significantly higher [ 64 Cu]W The biodistribution study showed a significant decrease in L12 uptake after AtzMab treatment (Figure 35G). Compared with conventional tumors, 64 Cu] 77% of WL12 bonds The tumor PD by AtzMab was reduced (Figure 35H, Figure 36D) (P<0.0001). We demonstrated that PD-L1 targeting was measured 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 (Figure 35I). 64 Cu]WL12-PET detects graded levels of PD-L1 expression in tumors A single 20 mg / kg dose of AtzMab was shown to significantly increase PD-L1 levels in tumors. This demonstrates that the serotonin receptor can be involved in the

[0124] 3.3.3. Quantification of dynamic changes in PD-L1 expression. PD-L1 is involved in the expression of various cytokines. Interferon, in particular, contributes to the dynamic and spatiotemporal heterogeneity in PD-L1 expression. It is known that it is upregulated in response to interferon gamma (IFNγ) (Taube et al., 2013). 015; Taube et al., 2012). We evaluated the robustness of quantifying [64Cu]WL12 and its effect on the AtzMab treatment. Blockade of such upregulated PD-L1 [ 64 Cu]WL12-PET It was determined whether the stimuli could be used in the same manner as in the control group (Figures 37A, 37B, 37C, 37D, 37E, and Figure 37F).

[0125] To do so, we analyzed A549 NSCL with doxycycline-inducible PD-L1 expression. A549 expressed PD-L1 at baseline. The Kras G12S lung adenocarcinoma cell line expresses low levels of the α-Kras gene. PD-L1 in the virus pINDUCER20 vector (Meerbrey et al., 2011) Transduce, select with G418, and confirm PD-L1 induction by flow cytometry ( (Figure 37A), was used in in vitro and in vivo studies. Binding of Cy5-PD-L1-mAb to phosphorylated A549-iPDL1 cells was significantly increased in WL12 The activity was blocked by WL12, demonstrating its specificity (Fig. 37B). 64 Cu WL12 incubation in doxycycline-treated vs. untreated cells and P D-L1 showed a 5.5-fold increase in radioactivity uptake in low A549 cells (P < 0.05). .0001) to 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 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 was increased by [ 64 Cu]WL12-PET and biodistribution studies As quantified by ab-treated group was reduced by >75% (Fig. 37D and Fig. 37E). IHC analysis of the tumors showed that A5 49-iPDL1 but not A549 tumors showed strong PD-L1 signals. The results confirmed the phasing 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. Therefore, 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 by different antibodies. Radiolabeled Anti-PD-L1 antibodies have been developed and are being used to treat human tumor xenografts and syngeneic mouse tumor models. Their potential to non-invasively 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. et al., 2015; Oosting et al., 2016), and faster clearance times (hours vs. days). ) is required (Wu, 2014). A further limitation is that Observations made with labeled antibodies are highly specific to the antibody under investigation and can reveal valency, shape, size, It is determined by antibody properties such as length, isoelectric point, and dosage, each of which affects its pharmacokinetics. Such unique biophysical characteristics of mAbs also affect plasma half-life, tissue exposure, and ultimately impact efficacy. (i) Target engagement of PD-L1 antibodies (ii) takes into account the properties 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 To quantify PD-L1 engagement in tumors non-invasively using 64Cu]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 FIG. 39D). In all treated mice, tumor Low signal, low levels of free PD-L1 from tumor PD-L1 engagement and Radiotracer blockade by the mAb was confirmed. Ex vivo quantification of tumors confirmed these We confirmed the findings of and demonstrated that 120 min after injection, [ 64 Cu]WL The uptake of 12 was demonstrated to be approximately 60% less compared to the saline control (Figure 39E). IHC analysis of saline controls showed moderate to high PD-L1 intensity in the tumors ( (Figure 39F). The results show that tumor PD-L1 engagement by PD-L1 therapeutic mAbs was Despite the different biophysical properties, plasma and tissue kinetics of each antibody, 64 Cu]WL It has been demonstrated that quantification can be achieved by 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 AtzMab and its mouse chimera (PRO3) target PD-L1 in human and colon tumors It was discovered that the accumulation of 04397) can be reduced (Chatterjee 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 a tumor model (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. Such effects also prevent the accumulation of mAbs (Goel et al., 2011). 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 was 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 and 2 h after injection of Cu]WL12 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 was not significantly different from that in tumors treated with AtzMab The PD-L1 occupancy rate was low (Figure 41A). There was a proportional decrease in signal intensity in the tumor at each dose, with a 3.2 mg / kg dose In this study, the antibody demonstrated nearly 100% target engagement with the tumor.

[0130] Radioactivity accumulated in the tumor (%ID / g) was then used to determine the inhibitory sigmoid E max The model was fitted. The %ID / g data were based on the AtzMab The dose and peptide radiotracer [ 64 Cu] in tumors detected using WL12 The relationship between the reduction in free PD-L1 ligand in the 14-HT100 / 1000 mice and the 14-HT100 / 1000 mice 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 in 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 IDs involved in 90% and 96% of 90 and ID 96 are 0.87 mg / These dose levels were equivalent to 1.19 mg / kg and 1.20 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 volume is 50 mL / kg, the ID 50 , ID 90 and ED 96 The expected mean plasma concentrations resulting from these are tentatively 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 is characterized by the fact that antibody binding stabilizes or internalizes PD-L1 and It is a small molecule that may affect the natural dynamics of PD-L1, including the development of therapeutic antibodies against PD-L1. Unlike the interactions of the antibodies, which can have a significant impact on the tumor and serum kinetics of the antibody (Tabr In early pharmacokinetic studies of AtzMab, the doses ranged from 0.6 to 1 mg / kg Nonlinear PK at doses below 1 mg / kg and linear PK at doses above 1 mg / kg have been reported, leading to ATA. A trend towards 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, we administered nonlinear and linear CT scans to NSG mice bearing MDAMB231 tumors, respectively. Injections of AtzMab at doses of 0.6, 10 or 20 mg / kg produced morphokinetic 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 significantly higher 64 Cu]WL12 uptake In contrast, in the 10 or 20 mg / kg treatment groups, 64 Cu] There was no significant difference in WL12 uptake over time. At 120 hours, 64 Cu]WL12 uptake was similar in the 0.06 mg / kg treated and saline control groups; This suggested clearance of the drug from the tumor and reflected the non-linear PK of AtzMab at low doses. Results show a dose- and time-dependent increase in PD-L1 engagement in mouse models. Both existential and non-existential changes 64 Cu]WL12-PET.

[0134] Discussion Immune checkpoint drugs are being tested in hundreds of clinical trials, of which approximately 25 % of patients receiving PD-L1 therapy have To respond to treatments, the molecular and cellular basis of response and resistance to these therapies has been investigated using transcriptional, genetic, and epigenetic studies. The relationship of dose to efficacy-related drug accumulation and target saturation 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 determine the tumor-specific expression of both tumor-intrinsic and tumor-extrinsic parameters. provides real-time PD-L1 saturation / occupancy data and is widely applicable This lack of knowledge has led to problems in dose selection, dose optimization, treatment development, and toxicity. In our current study, radiolabeled PD The PD-L1 binding peptide can non-invasively 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 antibody isotype, kinetics, ATA, and catabolism can be used to inhibit PD-L1:PD-1 interactions in tumors, It has been shown to provide a universal means for monitoring therapeutic activity in the body.

[0135] IHC-based clinical trials have been developed previously to assess PD-L1 expression in tumors Although it has been reported (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 be a potent inhibitor of immunotherapy responses. Such an approach has serious drawbacks because the response is inherently delayed, complex, and abscopal. In addition, 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 Such 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 issues are often impractical for immune checkpoints. 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 a PET radiotracer that can evaluate the PET signal 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-associated leukemia. It is quantifiable within the workflow and has important clinical implications for patient selection and monitoring treatment. Demonstrate that

[0136] PD-L1 therapeutic antibodies have become important drugs in cancer immunotherapy. For these drugs, in vitro binding affinity measurements and occupancy studies are useful for dose selection in CNS diseases. It is routinely used for the selection of markers and prediction of pharmacological response (Lee et al., 2006). However, large molecules such as antibodies can be identified based on in vitro binding affinity. This poses unique challenges in predicting receptor occupancy in vivo (Agor The antibody concentration in tumors is a function of antigen density and turnover, tumor burden, and tumor 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 affinity, dose, patient variability, and cachexia. It is further influenced by tumor-extrinsic factors such as tumor quality and the development of anti-therapeutic antibodies (She Existing PK / PD prediction models are limited in predicting optimal doses. 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 tumors in real time. We demonstrate that it is possible to measure PD-L1 occupancy by therapeutic antibodies.

[0137] Atezolizumab and other drugs 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. These measurements and mathematical modeling-derived occupancy predictions are often subject to the This is specific to this antibody and therefore does not limit the generalizability of such observations to other PD-L1 mAbs. The number of PD-L1 therapeutic options is expanding. Evaluate therapeutic mAbs for antibody kinetics and tumor target engagement There is a need for tools that can be used to improve the performance of a system. 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. This demonstrates that quantification of PD-L1 expression is possible for all PD-L1 therapeutic mAbs in clinical trials. This is a concept that can be applied to:

[0138] Taken together, the data disclosed herein provide insight into the dynamic changes in PD-L1 expression in tumors and in response to treatment. PD-L1 saturation / occupancy by targeted antibodies is independent of two features: antibody specificity and by considering the intrinsic and extrinsic parameters of the tumor, We demonstrate that the antibody-specific markers can be quantified in a quantitative manner using three different therapeutic antibodies: AtzMab, AveMab For DurMab, the results of this disclosure relating dose to PD-L1 occupancy in tumors are , which is expected to be related to treatment response and medication effect.

[0139] Overview. The subject matter of the present disclosure is that radiolabeled PD-L1 binding peptides are capable of detecting variable and dynamic PD-L1 expression levels. The level can be detected non-invasively and tumor intrinsic parameters (PD-L1 expression, , 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 to monitor the therapeutic activity of PD-L1 antibodies that block PD-1 interactions Demonstrate that

[0140] [ 64 Cu]WL12 study demonstrated variable and dynamic changes in PD-L1 expression can be quantified within standard clinical workflows and is an important tool for patient selection and monitoring treatment Demonstrate important clinical implications.

[0141] Existing PK / PD prediction models for antibodies use in vivo It relies on tro 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 it is possible to measure PD-L1 occupancy by antibodies.

[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 tro and in vivo data have demonstrated that it is possible to quantify PD-L1 saturation / occupancy in tumors, which has implications for 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, or other reference were incorporated by reference in its entirety. References are hereby incorporated by reference to the same extent as if each reference was specifically and individually indicated to be incorporated by reference. Numerous patent applications, patents, and other references are incorporated herein by reference. Although references are made to the above documents, such references are not intended to be construed as limiting the scope of the invention. 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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

[Claim 1] 1. An imaging agent comprising a conjugate of a peptide having binding specificity for Programmed Death Ligand 1 (PD-L1) and a reporting moiety, and optionally a linker, which, when present, links the peptide and the reporting moiety, or, when not present, the reporting moiety is directly attached to the peptide via a primary amine of an amino acid of the peptide, comprising the compound: 【Chemistry 1】

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