Imaging and targeting of programmed death ligand-1 (PD-L1) expression

Abstract

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

Description

[Technical Field]

[0001] Federal government-funded research or development This invention was made with the support of the U.S. government under the National Institutes of Health (NIH) designations CA236616 and CA166131. ​​The government has certain rights to this invention. background [Overview of the project] [Problems that the invention aims to solve]

[0002] Despite the effective application of immune checkpoint therapy (ICT) across a wide range of cancers, only a subset of patients with advanced cancer experience significant clinical response and survival. (Ribas and Wolchok, 2018). The challenge facing clinicians and researchers is how to get the most effective immunotherapy to patients as quickly as possible. Abundant clinical trial data is increasingly revealing that a single biomarker may not capture the range and breadth of clinical response to ICT. (Havel et al., 2019). Rather, the incorporation of multiple biomarker panels, including both pharmacodynamic and predictive biomarkers, is becoming necessary. (Havel et al., 2019). The number of tests that can be performed by baseline and intra-treatment biopsies is limited by the amount of biopsy tissue and has several drawbacks, including intertumor and intratumor heterogeneity and sampling errors. These problems are further exacerbated in difficult-to-access locations, as in lung and pancreatic cancers, and limit our ability to measure the pharmacodynamic effects of ICT. Imaging techniques, such as positron emission tomography (PET), enable repeated whole-body sampling and facilitate real-time quantification of pharmacodynamic effects. However, PET is not fully utilized in ICT guidance because of limited access to molecularly targeted radiotrackers that accurately report primarily on the activity of immune infiltration. Outline [Means for solving the problem]

[0003] In some embodiments, the subject matter of this disclosure provides imaging agents comprising a compound of formula (I): [ka] In the formula, L is a linker, which may or may not exist, and when it exists, it has the following general formula: [ka] In the formula: X is S or O; a, e, f, g, i, and j are integers independently selected from the group consisting of 0 and 1; b, d, h, and k are integers independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; c is an integer ranging from 0 to 40; each R1 is H or -COOR2, where R2 is H or C1-C4 alkyl; Ar is a substituted or unsubstituted aryl or heteroaryl; and A is a reporting molecule selected from the group consisting of chelating agents, radiolabeled substrates, fluorescent dyes, photoacoustic reporting molecules, and Raman-active reporting molecules, or a terminal group selected from the group consisting of -NR3R4 or C≡N, where R3 and R4 are independently selected from the group consisting of H and C1-C4 alkyl.

[0004] In other embodiments, the subject matter of the present disclosure provides an imaging method for detecting programmed death ligand 1 (PD-L1), the method comprising (a) providing an effective amount of imaging agent of formula (I); (b) contacting one or more cells or tissues with the imaging agent; and (c) creating an image for detecting PD-L1.

[0005] In another embodiment, the subject matter of the present disclosure provides a kit for detecting programmed death ligand 1 (PD-L1), the kit comprising an imaging agent of formula (I).

[0006] Certain aspects of the subject matter of this disclosure have been described above, but these are addressed in whole or in part by the subject matter of this disclosure, and other aspects will become apparent as the description progresses, in connection with the accompanying examples and figures, which are best described below. [Brief explanation of the drawing]

[0007] This patent or application file includes at least one drawing produced in color. A copy of the publication of this patent or patent application, along with the color drawing, will be provided by the office upon request and payment of the necessary fees.

[0008] Having thus described the subject matter of this disclosure in general terms, I now refer to the attached figures, which are not necessarily drawn to scale, and are as follows:

[0009] [Figure 1] Figures 1A, 1B, 1C, and 1D show the synthesis and in vitro characterization of [18F]DK222. Figure 1A shows the structure and schema for the preparation of [18F]DK222. Figure 1B demonstrates that DK221, DK222, and non-radioactive [19F]DK222 repress PD1:PD-L1 interactions at nanomolar concentrations in protein-based assays. Figure 1C is a flow cytometry histogram showing the gradient levels of PD-L1 expression in human TNBC, melanoma, and Chinese hamster ovary cells with stable human PD-L1 expression. Figure 1D demonstrates that the binding of [18F]DK222 to cells is PD-L1 expression-dependent and decreases in the presence of 1 μM of an unmodified peptide exhibiting specificity. Independent t-tests in Figure 1D showed ****, P<0.0001; NS, not significant; [Figure 2]Figures 2A, 2B, 2C, and 2D show the in vivo kinetics of [18F]DK222 in mice bearing TNBC xenografts. Figure 2A demonstrates high and specific uptake of [18F]DK222 in high-PD-L1-expressing MDAMB231 tumors, but not in mice receiving blocking doses or low-PD-L1-expressing SUM149 tumors (n=3-4). Whole-body volume-rendered PET-CT images of xenograft-bearing NSG mice were acquired at 15, 60, and 120 minutes after infusion of 200 mCi (7.4 MBq) of [18F]DK222. Blocking-dose mice received 50 mg / kg of unmodified peptide 30 minutes prior to radiotracer infusion. Figure 2B shows rapid accumulation and retention of [18F]DK222 in MDAMB231 tumors over several hours, which is not observed in SUM149 tumors or at blocking doses (n=4-5). Figure 2C is a time-activity curve obtained from in vivo distribution data showing rapid clearance of [18F]DK222 from circulation, as indicated by a high target-to-muscle (and blood) ratio (n=4-5). Data are derived from in vivo distribution studies shown in Table 1. Figure 2D shows IHC staining for PD-L1 in the corresponding tumors. By independent t-test of Figure 2C, ****, P<0.0001; NS, not significant; [Figure 3]Figures 3A, 3B, and 3C illustrate the high-contrast imaging of [18F]DK222 PET in mice bearing human melanoma xenografts at 60 minutes. Figure 3A shows high and specific uptake of [18F]DK222 in LOX-IMVI tumors expressing PD-L1, and not in mice receiving blocking doses or low-PD-L1 expressing MeWo tumors (n=3-4). Whole-body volume-rendered PET-CT images of xenograft-bearing mice were acquired 60 minutes after [18F]DK222 injection. Figure 3B shows tumor uptake of [18F]DK222 by ex vivo biodistribution in NSG mice bearing LOX-IMVI or MeWo tumors (n=5). Figure 3C shows IHC staining for PD-L1 in the corresponding tumors. Independent t-test in Figure 3B showed ****, P<0.0001; NS, not significant; [Figure 4]Figures 4A, 4B, 4C, 4D, 4E, 4F, and 4G demonstrate that [18F]DK222 uptake correlates with total PD-L1 levels in tumors induced by aPD-1 therapeutic agents. Figure 4A is a schematic diagram of the experiment. Figure 4B demonstrates that huPBMC mice with A375 melanoma tumors and treated with a single dose of 10 mg / kg of nivolumab or pembrolizumab for 7 days show increased [18F]DK222 uptake within the tumors. Representative images of three mice are shown in Figures 4B and 4C. Figure 4C illustrates that IHC analysis of tumor sections from imaging mice shows increased immunoreactivity for PD-L1 and CD3 in nivolumab and pembrolizumab-treated mice compared to Serene-treated controls and NSG mice. Figure 4D shows the uptake of [18F]DK222 in tumors quantified by its distribution in vivo (n=8-13). Figures 4E and 4F demonstrate that PD-L1 levels on tumors and immune cells (Figure 4E) and the number of CD45 cells analyzed by flow cytometry (Figure 4F) indicate the effects of different PD-1 antibodies. Figure 4G illustrates the strong correlation observed between [18F]DK222 uptake and total PD-L1 levels in the tumor microenvironment. Simple linear regression and Pearson coefficients in Figure 4G with 95% CI were obtained by one-way ANOVA in Figure 4D: ****P<0.0001;***, P<0.001;**, P<0.01. [Figure 5]Figures 5A, 5B, 5C, 5D, and 5E demonstrate that accessible PD-L1 levels quantified using [18F]DK222 indicate dose-dependent PD-L1 engagement by atezolizumab. Figure 5A shows that [18F]DK222 enables in vitro quantification of accessible PD-L1 levels in the presence of aPD-L1 mAbs; Figure 5B is a schematic diagram of the experiment. Figures 5C and 5E demonstrate that reduced uptake of [18F]DK222 is observed in LOX-IMVI tumors with increased atezolizumab doses. NSG mice were treated with different doses of atezolizumab for 24 hours prior to [18F]DK222 infusion. Whole-body volume-rendered PET-CT images of mice were obtained 60 times after injection of [18F]DK222 (Figure 5D) (n=3) and ex vivo in vivo distribution (Figure 5E) (n=5). One-way ANOVA and Tukey's multiple comparisons test in Figure 5E yielded the following results: ****, P<0.0001, **, P<0.01; [Figure 6] Figures 6A, 6B, 6C, and 6D show the pharmacological activity of PD-L1 therapeutic agents quantified in tumors using [18F]DK222-PET. Figure 6A is a schematic diagram of the experiment. Figure 6B shows that [18F]DK222 uptake in LOX-IMVI tumors of mice treated with 1 mg / kg antibody for 24 and 96 hours captures different PD-L1 occupancy and PK in antibody-affinity-dependent tumors (n=3). NSG mice were treated with atezolizumab, avelumab, or durvalumab at a dose of 1 mg / kg for 24 and 96 hours prior to [18F]DK222 injection. Nivolumab was used at 1 mg / kg and Serene was used as a control. Whole-body volume-rendered PET / CT images of mice were acquired 60 minutes after [18F]DK222 injection. Figures 6C and 6D show the uptake of [18F]DK222 in tumors quantified by in vivo distribution in LOX-IMVI (Figure 6D, n=8-19) and MDAMB231 (Figure 6D, n=7-18) tumor-carrying mice; [Figure 7]Figure 7A shows [18F]DK222 PET in a non-human primate (Papio anubus). Papio anubus were injected with ~(approximately) 5 mCi of [18F]DK222 using PET imaging, and whole-body images were acquired at different time points. The PET images showed significant radioactivity uptake in the bladder, kidneys, and spleen. Interestingly, high uptake was also observed in what appeared to be lymph nodes; [Figure 8] Figures 8A, 8B, 8C, and 8D demonstrate that [18F]DK222 PET in mice with human lung cancer xenografts shows high contrast images at 60 minutes. Figure 8A shows PD-L1 expression levels in lung cancer analyzed by flow cytometry. Figure 8B shows in vitro uptake of [18F]DK222 in lung cancer cell lines. Figure 8C shows high and specific uptake of [18F]DK222 in H2444 tumors expressing PD-L1, and not in mice with A549 tumors expressing low PD-L1 (n=3-4). Whole-body volume-rendered PET-CT images of xenograft-bearing mice were acquired 60 minutes after [18F]DK222 injection. Figure 8D shows the tumor uptake of [18F]DK222 by ex vivo in vivo distribution in NSG mice with H2444, H226, and A549 tumors (n=5); [Figure 9]Figures 9A, 9B, 9C, and 9D demonstrate that [18F]DK222 PET in mice with human bladder cancer xenografts produces high-contrast images at 60 minutes. Figure 9A shows PD-L1 expression levels in bladder cancer cell lines analyzed by flow cytometry. Figure 9B shows in vitro uptake of [18F]DK222 in bladder cancer cells with fluctuating PD-L1 expression. Figure 9C shows high and specific uptake of [18F]DK222 in BFTC909 tumors expressing PD-L1, and not in mice with SCaBer tumors with low PD-L1 expression (n=3-4). Whole-body volume-rendered PET-CT images of xenograft-bearing mice were acquired 60 minutes after [18F]DK222 injection. Figure 9D shows the tumor uptake of [18F]DK222 by ex vivo in vivo distribution in NSG mice with BFTC909, T24, and SCaBER tumors (n=5); [Figure 10] This is a schematic diagram of the structure of DK221 and the synthesis of [19F]DK222; [Figure 11] Figures 11A, 11B, 11C, and 11D are as follows: Figure 11A, reverse-phase HPLC chromatogram of DK222; Figure 11B, ESI-MS of DK222; Figure 11C, reverse-phase HPLC chromatogram of [19F]DK222; Figure 11D, ESI-MS of [19F]DK222; [Figure 12] [18F] This is a schematic diagram for the synthesis of DK222; [Figure 13] Figures 13A, 13B, and 13C are as follows: Figure 13, reverse-phase HPLC chromatogram of the crude reaction mixture of [18F]DK222; Figure 13B, radiochemical purity of [18F]DK222; Figure 13C, chemical identity of [18F]DK222; [Figure 14] This demonstrates the stability of compounded [18F]DK222; [Figure 15]Figures 15A, 15B, and 15C show the following: Figure 15A, Effect of non-radioactive DK221 carrier on [18F]DK222 uptake in MDAMB231 and SUM149 tumors. Co-infusion of variable doses of DK221 and [18F]DK222 shows a decrease in radioactivity uptake with increasing carrier dose in PD-L1-positive MDAMB231 tumors, but not in PD-L1-negative SUM149 tumors. Figure 15B, In vivo distribution data showing mean %ID / g values ​​with 95% confidence intervals. Figure 15C, Carrier dose has minimal effect on [18F]DK222 uptake in selective tissues. Uptake in the 30 μg dose group is consistently high in all tissues for reasons unknown; [Figure 16] This study shows the ex vivo distribution of [18F]DK222 in mice carrying LOX-IMVI and MEWO melanoma tumor xenografts. Mice received 50 μCi of [18F]DK222, and tissue was collected 60 minutes later. The data shown are mean ± SEM (n=4-5 / group). ****, P<0.0001 by independent t-test; [Figure 17] This demonstrates the effect of IFNγ treatment on PD-L1 levels, as evaluated by flow cytometry, in melanoma cell lines. [Figure 18] This shows the ex vivo in vivo distribution of [18F]DK222 in selected tissues in huPBMC mice carrying A375 xenografts and treated with aPD-1 mAbs. Mice received 50 μCi of [18F]DK222, and tissues were collected 60 minutes later. [Figure 19] This shows the ex vivo in vivo distribution of [18F]DK222 in selected tissues in NSG mice carrying LOX-IMVI xenografts and treated with atezolizumab at doses of 0.3 mg / kg and 20 mg / kg. Mice received 50 μCi of [18F]DK222, and tissues were collected 60 minutes later. [Figure 20]This shows the ex vivo distribution of [18F]DK222 in selected tissues in NSG mice carrying LOX-IMVI xenografts and treated with 1 mg / kg doses of aPD-L1 mAbs for 24 and 96 hours; [Figure 21] This is a MALDI-TOF MS for the DK222; [Figure 22] This is the ESI-MS for the DK331; [Figure 23] This is MALDI-MS for the DK331; [Figure 24] This is the ESI-MS for the DK225; [Figure 25] This is MALDI-MS for the DK223; [Figure 26] This is MALDI-MS for the DK385; [Figure 27] This is the ESI-MS for the DK254; [Figure 28] This is the ESI-MS for the DK265; [Figure 29] This is ESI-MS for DK365; [Figure 30] This is the ESI-MS for the DK360; [Figure 31] This is the MALDI-TOF for the DK388; [Figure 32] This is RP-HPLC of crude [18F]PyTFP; [Figure 33] This is RP-HPLC of crude [18F]DK221Py; [Figure 34] This is RP-HPLC of pure [18F]DK221Py; [Figure 35] This is an in vivo evaluation of [18F]DK221Py in hPD-L1 / CHO; [Figure 36] Data from HTRF PD1 / PD-L1 binding assays for DK221, DK222, and DK291 ([19F]DK222) are shown; and [Figure 37] Data from HTRF PD1 / PD-L1 binding assays for DK225, DK223, DK385, and DK331 are shown.

[0010] Detailed description [Modes for carrying out the invention]

[0011] The subject matter of this disclosure is described more fully below with reference to the accompanying drawings, which illustrate some, though not all, embodiments, of the subject matter of this disclosure. Similar numbers refer to similar elements throughout. The subject matter of this disclosure can be embodied in many different forms and should not be construed as being limited to the embodiments described herein; rather, these embodiments are provided so as to satisfy the applicable legal requirements of this disclosure. In fact, many modifications and other embodiments of the subject matter of this disclosure described herein will be conceivable to a person skilled in the art (also known as a person skilled in the art) who has an interest in the teachings shown in the foregoing description and the accompanying drawings. It is understood that the subject matter of this disclosure is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be within the scope of the accompanying claims. I. Imaging and targeting of programmed death ligand-1 (PD-LI) expression

[0012] The tools currently available for quantifying systemic immune responses are limited. The subject of this disclosure is, in part, directed towards the development of radiopharmaceuticals for programmed death ligand-1 (PD-LI), the most widely used biomarker for selecting patients for immune checkpoint therapy (ICT), and which has proven useful in predicting responses to ICT in several cancers. Garon et al., 2015; Reck et al., 2016; Reck et al., 2019; Herbst et al., 2019; Hellmann et al., 2018; Peters et al., 2019; Spigel et al., 2019; Yarchoan et al., 2019; Melosky et al., 2018. For this reason, peptide-based radiopharmaceuticals and analogues have been developed to measure PD-L1 levels in order to predict the efficacy of ICT in real time.

[0013] More specifically, the subject of this disclosure provides a highly specific peptide-based positron-emitting tomography (PET) imaging agent that can detect PD-L1 expression in tumor and immune cells immediately after injection of a radiotracer. The imaging agent of this disclosure fits within and is not limited to a standard clinical workflow of imaging within 60 minutes of administration, but is applicable to imaging various types of cancer, infections, and inflammatory entities, including experimental models of chronic bacterial infections, disseminated tuberculosis, lupus, and rheumatoid arthritis. A. Composition containing imaging agents

[0014] In some embodiments, the subject of this disclosure provides imaging agents comprising a compound of formula (I): [ka] In the formula, L is a linker, which may or may not exist, and when it exists, it has the following general formula: [ka] In the formula: X is S or O; a, e, f, g, i, and j are integers independently selected from the group consisting of 0 and 1; b, d, h, and k are integers independently selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; c is an integer ranging from 0 to 40; each R1 is H or -COOR2, where R2 is H or a C1-C4 alkyl group; Ar is a substituted or unsubstituted aryl or heteroaryl group; and A is a reporting moiety selected from the group consisting of chelating agents, radiolabeled substrates, fluorescent dyes, photoacoustic reporting molecules, and Raman-active reporting molecules, or a terminal group selected from the group consisting of -NR3R4 or C≡N, where R3 and R4 are independently selected from the group consisting of H and a C1-C4 alkyl group.

[0015] As used herein, C1-C4 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. The term "aryl" means an aromatic hydrocarbon substituent that can be a single ring or multiple rings (e.g., one to three rings, etc.) unless otherwise stated, and they They are fused to each other or covalently bonded. The term "heteroaryl" refers to an aryl group (or ring) containing one to four heteroatoms selected from N, O, and S (in each separate ring, if there are multiple rings), where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms (plural) are optionally quaternized.

[0016] In some embodiments, the linker is selected from the group consisting of: [ka] Therefore, p is an integer chosen from 0, 1, 2, 3, and 4; [ka] Therefore, q is an integer chosen from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; [ka] Therefore, r is an integer chosen from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; [ka] Therefore, s is an integer in the range of 1 to 40, and t is an integer chosen from 0 or 1. It is a number; [ka] Therefore, s is an integer in the range of 1 to 40, and t is an integer chosen from 0 or 1; and [ka] Therefore, s is an integer in the range of 1 to 40, and t is an integer chosen from 0 or 1.

[0017] A person skilled in the art will recognize, in considering the subject matter of this disclosure, that various combinations of chelating agents / radioactive metal ions are suitable for use with the imaging agents of this disclosure. Representative chelating agents are known in the art. In non-limiting examples, certain chelating agents and linkers are disclosed in U.S. Patent Application Publication Nos. 2015 / 0246144 and 2015 / 0104387, each of which is incorporated herein by reference as a whole.

[0018] In some embodiments, the reporting moisture is a chelating agent, and the chelating agent is DOTAGA(1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecane-1,7,4,7,10-tetraacetic acid), DOTA-tris(t-butyl) ester, DOTAGA-(t-butyl)4, DOTA-di(t-butyl) ester, DOTASA(1, 4,7,10-Tetraazacyclodecane-1-(2-succinic acid)-4,7,10-triacetic acid), CB-DO2A(10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane), DEPA(7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododeca-1-ylacetic acid), 3p-C-DEPA(2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-to Ris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]pentan-2-yl)amino]acetic acid), TCMC(2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmethyl)-cyclododecane), oxo-DO3A(1-oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid), DO3A-(t-butyl), DO3AM(2,2',2"-( 1,4,7,10-Tetraazacyclododecane-1,4,7-Tolyl(triacetamide), p-NH2-Bn-Oxo(Oxo)-DO3A(1-Oxa-4,7,10-Tetraazacyclododecane-5-S-(4-aminobenzyl)-4,7,10-triacetic acid), TE2A((1,8-N,N'-bis-(carboxymethyl)-1,4,8,11-Tetraazacyclotetradecane), MM-TE2A, DM-TE2A, CB-TE2A(4,11-bis(carboxymethyl)-1,4,8,11-Tetraaza Bicyclo[6.6.2]hexadecane), CB-TE1A1P (4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid), CB-TE2P (1,4,8,11-tetraazacyclotetradecane-1,8-bis(methanephosphonic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), NOTA (t-butyl)2, NO 2A(1,4,7-Triazacyclononane-1,4-bis(acetic acid)-7-(acetamide), NODA(1,4,7-Triazacyclononane-1,4-diaacetate), NODAGA(1,4,7-Triazacyclononane,1-glutaric acid-4,7-acetic acid), NODAGA(t-butyl)3, NOTAGA(1,4,7-Triazonanane-1,4-diyl)diacetic acid), DFO(Desferoxamine), DTPA(2-[bis[2-[bis(carboxymethyl) [Amino]ethyl]amino]acetic acid), DTPA-tetra(t-butyl) ester (diethylenetriamine-N,N,N"-tetra-tert-butylacetate-N'-acetic acid), NETA ([4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl}acetic acid), TACN-TM (N,N',N", tris(2-mercaptoethyl)-1,4,7-triazacyclononane), Diamsar (1,8-di Amino-3,6,10,13,16,19-Hexazabicyclo(6,6,6)eicosane,3,6,10,13,16,19-Hexazabicyclo[6.6.6]eicosane-1,8-diamine), Sarar (1-N-(4-aminobenzyl)-3,6,10,13,16,19-Hexazabicyclo[6.6.6]eicosane-1,8-diamine), AmBaSar (4-((8-amino)-3,6,10,13,16,19-Hexazabicyclo[6.6.6) Eicosan-1-ylamino)methyl)benzoic acid), BaBaSar (BaBaSar), Tris(hydroxypyridinone)(THP), THP(benzyl)3, NOPO(3-(((4,7-bis((hydroxy(hydroxymethyl)phosphoryl)-methyl)-1,4,7-triazonan-1-yl)methyl)(hydroxy)phosphoryl)propanoic acid), TRAP(3,3',3"-(((1,4,7-triazonan-1,4,7-tolyl)tris Selected from the group consisting of (methylene))tris(hydroxyphosphoryl))-tripropanoic acid), p-NH2-Bn-PCTA(3,6,9,15-tetrazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-aminobenzyl)-3,6,9-triacetic acid), and biotin (5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazole-4-yl]pentanoic acid).

[0019] In some embodiments, the chelating agent is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0020] In some embodiments, the chelating agent is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), NODA (1,4,7-triazacyclononane-1,4-diacetate); NODAGA (1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid), and biotin (5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid).

[0021] In some embodiments, the reporting moiety is a chelating agent, and the chelating agent further comprises a radioactive metal selected from the group consisting of 94m Tc, 99m Tc, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 55 Co, 57 Co, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Pb, 153 Sm, 166 Ho, 152 Gd, 82 Rb, 89 Zr, 166 Dy, and Al 18 and further comprises a radioactive metal selected from the group consisting of F.

[0022] In some embodiments, the substrate is, for example, based on the chelation of aluminum fluoride using the AlF method based on the chelation of aluminum fluoride by NOTA, NODA, or other suitable chelating agents known in the art 18Labeled with F. For example, Liu S. et al., "One-step radiosynthesis of 18 F-AlF-NOTA-RGD2 for tumor angiogenesis PET imaging 18 "One-step radiosynthesis of F-AlF-NOTA-RGD2". Eur J Nucl Med Mol Imaging (European Journal of Nuclear Medicine and Molecular Imaging). 2011, 38(9):1732-41; McBride WJ et al., "A novel method of 18 F radiolabeling for PET 18 Novel method for radiolabeling of fluorine (F). J Nucl Med. (The Journal of Nuclear Medicine) 2009;50:991-998; McBride WJ, D'Souza CA, Sharkey RM, Karacay H, Rossi EA, Chang CH, Goldenberg DM. Improved 18 F labeling of peptides with a fluoride-aluminum-chelate complex 18 (F-labeling). See Bioconjug Chem. (Bioconjugate Chemistry) 2010;21:1331-1340.

[0023] A person of ordinary skill in this art will recognize that in some embodiments, the linker, "L" in formula (I), is absent, and the chelating agent is conjugated with DK221 through a linker moiety, which is part of the chelating agent as supplied. For example, in certain embodiments, as provided in Example 1 below, the lysine ε-amine of DK221 is used for bifunctional chelating agent conjugation using the NHS ester method. For example, if the chelating agent is NCS-MP-NODA(2,2'-(7-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid as supplied, the isothiocyanatobenzyl moiety is the linker between the NODA chelating agent and the lysine ε-amine of DK221.

[0024] Other linker moieties that may contain chelating agents as supplied include, but are not limited to, maleimide, NHS esters, anhydrides, NCS, NCS-benzyl, NH2-PEG, BCN, -NH2, propargyl, acetic acid, glutamic acid, and other similar substances.

[0025] In some embodiments, the reporting moiety is a radiolabeled substrate, and the radiolabeled substrate contains, 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, 19 F, 18 F, and 211 It contains radioactive isotopes selected from the group consisting of At.

[0026] In some embodiments, the radiolabeled substrate is: 18 F-labeled substrate or18 It contains an F-labeled substrate.

[0027] In some embodiments, 19 F-labeled substrate or 18 The fluorine-labeled substrates are 2-fluoro-PABA and 3 Selected from the group consisting of -fluoro-PABA, 2-fluoro-mannitol, N-succinimidyl-4-fluorobenzoate, and 2-pyridyl.

[0028] In some embodiments, the reporting moisture is a fluorescent dye, and the fluorescent dye is selected from the group consisting of: carbocyanine, indocarbocyanine, oxacarbocyanine, thuicarbocyanine, merocyanine, polymethine, coumarin, aminomethylcoumarin acetate (AMCA), rhodamine, tetramethylrhodamine (TRITC), xanthene, fluorescein, FITC, borondipyromethane (BODIPY) dye, Cy3, Cy5, Cy5. 5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor-350, AlexaFluor-405, AlexaFluor-488, AlexaFluor-546, AlexaFluor-555, AlexaFluor-594, AlexaFluor-633, AlexaFluor-647, AlexaFluor-660, AlexaFluor-680, AlexaFluor 700, AlexaElectro750, AlexaElectro790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight350, DyLight405, DyLight488, DyLight547, DyLight550, DyLight594, DyLight633, DyLight647, DyLight650, DyLight680, DyLight755, DyLight800, HiLyte These include Fluor (Highlight Fluor) 647, HiLyte Fluor 680, HiLyte Fluor 750, IR Dye 800, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, Cascade Blue, and Texas Red.

[0029] In some embodiments, the reporting molecule is a photoacoustic reporting molecule, which is selected from the group consisting of dyes or nanoparticles.

[0030] In some embodiments, the dye includes a fluorescent dye. In some embodiments, the fluorescent dye is selected from the group consisting of indocyanine-green (ICG), Alexa Fluor 750, Evans Blue, BHQ3, QXL680, IRDye880CW, MMPSense 680, Methylene Blue, PPCy-C8, and Cypate-C18.

[0031] In some embodiments, the nanoparticles include plasmonic nanoparticles, quantum dots, nanodiamonds, polypyrrole nanoparticles, copper sulfide nanoparticles, graphene nanosheets, iron oxide-gold core-shell nanoparticles, Gd2O3 nanoparticles, single-walled carbon nanotubes, and dye-loaded perfluorocarbon nanoparticles. Selected from the group consisting of nanoparticles and superparamagnetic iron oxide nanoparticles.

[0032] In some embodiments, the reporting molecule is a Raman-active reporting molecule, which is selected from the group consisting of single-walled carbon nanotubes (SWNTs) and surface-enhanced Raman scattering (SERS) agents.

[0033] In some embodiments, the SERS agent includes metal nanoparticles labeled with a Raman-active reporter molecule.

[0034] In some embodiments, the Raman-active reporter molecule includes a fluorescent dye. In some embodiments, the fluorescent dye is selected from the group consisting of Cy3, Cy5, rhodamine, and chalcogenopyryllium dyes.

[0035] In some embodiments, the imaging agent of formula (I) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] .

[0036] In some embodiments, imaging agents are capable of detecting PD-L1 in vitro, in vivo, and / or ex vivo. In some embodiments, imaging agents are capable of detecting PD-L1 in vivo. PD-L1 is expressed by various tumors, and its overexpression is induced in tumor cells as an adaptive mechanism in response to tumor-infiltrating cytotoxic T cells. A person with ordinary skill in the art will recognize that PD-L1 may include modifications and / or mutations, and is still applicable to the methods of the present disclosure as long as it can still be detected by the imaging agents of the present disclosure.

[0037] In some embodiments, the imaging agent of the present disclosure suppresses the interaction of PD-L1 and its ligand with programmed cell death protein 1 (PD-1). 50 It has a range of approximately 100 nM to approximately 1 pM. In some embodiments, IC 50 In one embodiment, the wavelength is less than 100 nM; in another embodiment, less than 10 nM; in yet another embodiment, less than 8 nM; in yet another embodiment, less than 5 nm; in yet another embodiment, less than 4 nm; and in yet another embodiment, less than 3 nM.

[0038] The term "bonding affinity" is a property that describes how strongly two or more compounds associate with each other in a non-covalent bond. Bonding affinity can be expressed qualitatively (e.g., "strong," "weak," "high," or "low") or quantitatively (e.g., K d It can be characterized by things like measurements. B. Detection method using imaging agents

[0039] In some embodiments, the subject matter of this disclosure provides a method for detecting immune checkpoint proteins, such as PD-L1. In some embodiments, the subject matter of this disclosure provides a method for detecting diseases, disorders, or conditions that result in overexpression of PD-L1, such as cancer, inflammation, infection, and other similar conditions.

[0040] Accordingly, in some embodiments, the subject of the present disclosure provides an imaging method (also referred to as an imaging technique) for detecting programmed death ligand 1 (PD-L1), the method comprising: (a) providing an effective amount of imaging agent of formula (I); (b) contacting one or more (also referred to as one or more) cells or tissues with the imaging agent; and (c) creating an image for detecting PD-L1.

[0041] As used herein, the terms “imaging” or “making an image” refer to the use of any imaging technique to visualize a detectable compound by measuring the energy released by the compound. In some embodiments, the term “imaging” refers to the use of any imaging technique to visualize by measuring the energy released by a compound after administration of the detectable compound to a subject, and after localization of the compound following administration. In some embodiments, the imaging technique involves administering a compound that can be detected externally to the subject. In some embodiments, the image is generated by differences in the spatial distribution of the imaging agent accumulating at various locations in the subject. In some embodiments, the administration of the imaging agent is done by injection.

[0042] The term “imaging agent” is intended to include, for example, compounds capable of imaging by positron emission tomography (PET). As used herein, “positron emission tomography imaging” or “PET” incorporates positron emission tomography imaging systems or equivalents and all devices capable of positron emission tomography imaging. The methods of the subject matter of this disclosure can be practiced using any such device, or variations of PET devices or equivalents, or in combination with any known PET methodology. This is possible. For example, see U.S. Patents No. 6,151,377; No. 6,072,177; No. 5,900,636; No. 5,608,221; No. 5,532,489; No. 5,272,343; No. 5,103,098, each of which is incorporated herein by reference. Animal Imaging Modality This includes imaging modalities, an example of which is microPETs (Corcorde Microsystems, Inc.).

[0043] Depending on the reporting moiety, the imaging agents of this disclosure can be used in PET, single-photon emission computed tomography (SPECT), near-infrared (fluorescence), photoacoustic, and Raman imaging.

[0044] In some embodiments, imaging involves scanning the entire subject or patient, or a specific area of ​​the subject or patient, using a detection system, and detecting a signal. The detected signal is then converted into an image. The resulting image should be read by an experienced observer, such as a physician. Typically, imaging is performed between approximately 1 minute and 48 hours after administration of the imaging agent. The precise timing of imaging will depend on factors such as the clearance rate of the administered compound, as will be readily apparent to those skilled in the art. The imaging timeframe may vary based on the radionucleotide used. In certain embodiments, imaging is performed between approximately 1 minute and 4 hours after administration, for example, between 15 and 30 minutes, between 30 and 45 minutes, between 45 and 60 minutes, between 60 and 90 minutes, and between 60 and 120 minutes. In some embodiments, PD-L1 is detected immediately, approximately 60 minutes after administration of the imaging agent to the subject. In some embodiments, imaging may be performed 24 hours after injection with a Zr-89 labeled peptide. In some embodiments, imaging may be performed 24 hours after injection with an I-124 labeled peptide.

[0045] Once an image is obtained, a person skilled in the art can determine the location of the compound. Using this information, the skilled person can determine, for example, whether a condition such as infection, inflammation, or cancer is present, the extent of that condition, or the effectiveness of the treatment the subject is receiving.

[0046] In some embodiments, contacting cells or tissues with imaging agents is performed in vitro, in vivo, or ex vivo. “Contacting” means any action that results in physical contact between at least one imaging agent of the subject matter of this disclosure and at least one cell or tissue. Thus, it may include exposing cells(plural) or tissue(plural) to imaging agents in an amount sufficient to result in contact between at least one imaging agent and at least one cell or tissue. In some embodiments, the method can be practiced in vitro or ex vivo by introducing the imaging agent and cells or tissue in a controlled environment, such as a culture dish or tube, and preferably by mixing them. In some embodiments, the method can be practiced in vivo, in which case exposure of at least one cell or tissue in a subject to at least one imaging agent of the subject matter of this disclosure by contact means, for example, by applying the imaging agent to the subject via any suitable route. In some embodiments, contacting cells or tissues with imaging agents is performed in the subject.

[0047] The term "effective amount" of imaging agent refers to the amount necessary or sufficient to provide a readable signal during imaging using positron emission tomography (PET), as described herein. The effective amount can vary depending on factors such as the size and weight of the subject, the type of disease, or the specific compound. For example, the selection of the compound can affect what constitutes the "effective amount." Those with ordinary skill in this art are included herein. By studying the contributing factors, it will be possible to determine the effective amount of the compound without excessive experimentation.

[0048] While the subjects to be diagnosed or treated by the methods of this disclosure in many of their embodiments are preferably human subjects, it is understood that the methods described herein are effective with respect to all vertebrate species intended to be included in the term “subject.” Thus, “subject” may include human subjects for medical purposes, such as the diagnosis or treatment of existing diseases, disorders, or conditions, or animal subjects for medical, veterinary, or developmental purposes. Preferred animal subjects include, but are not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and other of the same kind; bovines, e.g., cattle, oxen and other of the same kind; ovines, e.g., sheep, e.g., sheep and other of the same kind; caprines, e.g., goats and other of the same kind; and porsines, e.g., pigs, hogs and other of the same kind. Mammals are included, including: equines (animals of the Equidae family), examples of which are horses, donkeys, zebras, and other similar species; felines (animals of the Felidae family), including wild cats and domestic cats; canines (animals of the Canidae family), including dogs; lagomorphs (animals of the Lagomorpha order), including rabbits, hares, and other similar species; and rodents, including mice, rats, guinea pigs, and other similar species. Animals may also be transgenic animals. In some embodiments, subjects are human, but are not limited to, fetuses, neonates, infants, juveniles, and adult subjects. Furthermore, “subject” may include patients who are suffering from or suspected of suffering from a disease, disorder, or condition. Thus, the terms “subject” and “patient” are used interchangeably here. The subjects also include animal disease models (e.g., rats or mice used in experiments, and other similar types).In some embodiments, the subjects are humans, rats, mice, cats, dogs, horses, sheep, cattle (cows), monkeys, birds, or amphibians.

[0049] In general, imaging agents of the present disclosure can be administered to a subject for the detection of disease, disorder, or condition by any suitable route of administration, including orally, intranasally, intraocularly, rectally, vaginally, or parenterally; intravenously, intramuscularly, subcutaneously, intrathecally, including intrathecally, directly into the ventricle, intraarticularly, intrasternally, intrasynovally, intrahepatically, intracranially, intraperitoneally, intranasally, or intraocularly, into the cisterna magnus; topically, as a powder, ointment, or drop (including eye drops); percutaneously, via inhalation spray, or by other forms of delivery known in the present art, including buccally and sublingually.

[0050] The phrases “systemic administration,” “administer systemically,” “peripheral administration,” and “peripherally administration” as used herein mean the administration of a composition that enters the system of the subject or patient and is therefore subject to metabolism and other similar processes, such as subcutaneous or intravenous administration.

[0051] As used herein, the terms “parenteral administration” and “administer parenterally” mean, and are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrastemal injections and infusions.

[0052] In some embodiments, the imaging agent exhibits a target-to-non-target ratio of at least 3:1. In some embodiments, the term “target” refers to cells or tissues that exhibit overexpression of the PD-L1 protein, and the term “non-target” refers to cells or tissues that do not exhibit overexpression of the PD-L1 protein.

[0053] In some embodiments, imaging methods are used to detect cancer. “Cancer” in a subject or patient refers to the presence of cancerous cells, e.g., cells possessing uncontrolled proliferation, loss of differentiated function, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rates, and characteristic features of certain distinctive morphology and cellular markers. Under some circumstances, cancer cells are tumor forms; such cells may reside locally within an animal or circulate in the bloodstream as independent cells, e.g., leukemia cells. The cancers used herein include newly diagnosed or recurrent cancers and, without limitation, include blastomas, carcinomas (carcinomas), gliomas, leukemias, lymphomas, melanomas, myelomas, and sarcomas. As used herein, cancer includes head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer (such as triple-negative breast cancer), prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, digestive cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, brain cancer, and adenoma. In some embodiments, cancer includes stage 0 cancer. In some embodiments, cancer includes stage 1 cancer. In some embodiments, cancer includes stage 2 cancer. In some embodiments, cancer includes stage 3 cancer. In some embodiments, cancer includes stage IV cancer. In some embodiments, cancer is refractory and / or metastatic.

[0054] As used herein, “tumor” refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. As used herein, “solid tumor” is generally an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors may be found in the brain, colon, chest, prostate, liver, kidneys, lungs, esophagus, head and neck, ovaries, cervix, stomach, colon, rectum, bladder, uterus, testes, and pancreas, in non-limiting examples. In some embodiments, imaging methods are used to detect solid tumors. In other embodiments, imaging methods are used to detect metastatic cancer.

[0055] In some embodiments, imaging methods are used to detect infections. Infectious diseases, such as infections caused by any fungus or bacteria, are intended to be detected using the subject matter of this disclosure. As used herein, the term “infection” refers to the invasion of a host organism’s tissues by disease-causing organisms, their proliferation, and the host tissue’s response to these organisms and the toxins they produce. Infections include, but are not limited to, hospital-acquired infections, surgical infections, and severe abdominal infections, such as peritonitis, pancreatitis, gallbladder empyema, and pleura empyema, and bone infections, such as osteomyelitis. Detection of septicemia, sepsis and septic shock, immunosuppressants, cancer chemotherapy, radiation, contaminated IV fluids, hemorrhagic shock, ischemia, trauma, cancer, immunodeficiency, viral infections, and diabetes, or infections after use, are also intended. Examples of microbial infections, such as infections caused by bacteria and / or fungi, include Mycobacterium tuberculosis, E. coli, Klebsiella sp., Enterobacter sp., Proteus sp., Serratia marcescens, and Pseudomonas aeruginosa. This category includes Monas eruginosa (Pseudomonas aeruginosa), Staphylococcus spp. (Staphylococcus species, Staphylococcus species), including S. aureus (Staphylococcus aureus, Staphylococcus aureus) and coagulase-negative Staphylococcus (coagulase-negative Staphylococcus), Enterococcus sp. (Enterococcus species), Streptococcus pneumoniae (Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus influenzae, Bacteroides spp. (Bacteroides species), Acinetobacter spp. (Acinetobacter species), Helicobacter spp. (Helicobacter species), Candida sp. (Candida species), and others. Infections caused by resistant microorganisms include, for example, methicillin-resistant Staphylococcus. This includes methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). In some embodiments, the infection is a bacterial infection. In some embodiments, the infection is a chronic bacterial infection. In some embodiments, the bacterial infection is tuberculosis. In some embodiments, the infection is disseminated tuberculosis. In some embodiments, the infection may be hepatitis A, hepatitis B, hepatitis C, and / or human immunodeficiency virus.

[0056] In some embodiments, imaging methods are used to detect inflammation. Examples of disorders associated with inflammation include, but are not limited to, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, lupus, systemic lupus erythematosus, and vasculitis. In some embodiments, inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus.

[0057] PD-L1 binds to its receptor PD-1, which is found on activated T cells, B cells, and myeloid cells, to modulate activation or suppression. PD-L1 is also expressed on several immune cells, including macrophages. Therefore, the imaging agent of the present disclosure can be used to detect the expression of PD-L1 and to detect immune cells, such as T cells, B cells, and myeloid cells. In some embodiments, the imaging agent of the present disclosure detects immune cells in tumors. In some embodiments, the imaging agent of the present disclosure detects the systemic distribution of immune cells in a subject. In some embodiments, the imaging method is used to detect immune cell responses in infectious cells. In some embodiments, the imaging method is used to detect immune cell responses in inflammatory cells.

[0058] In some embodiments, imaging methods of the present disclosure detect and / or measure changes in PD-L1 expression, such as changes in PD-L1 expression induced by treatment. Such methods can be used to verify the effectiveness of a particular treatment method and / or to determine an effective therapeutic dosage range. C. Kits containing imaging agents

[0059] In some embodiments, the subject of the present disclosure provides a kit for detecting programmed death ligand 1 (PD-L1), the kit comprising an imaging agent containing a compound of formula (I), as described above.

[0060] Typically, a kit of the subject matter of this disclosure includes an imaging agent of the disclosure and instructions on how to carry out at least one method of the disclosure. The imaging agent is generally supplied in the kit in an amount sufficient to detect PD-L1 at least once in at least one subject or patient. The kit may also include some or all of the other reagents and supplies necessary to carry out at least one embodiment of the method of the disclosure.

[0061] In its simplest form, a kit according to the subject of this disclosure comprises a container containing at least one imaging agent according to the subject of this disclosure. In some embodiments, the kit comprises a plurality of containers, each of which may contain at least one imaging agent or other substances useful for carrying out one or more embodiments of the method of this disclosure.

[0062] The container may be of any material suitable for containing another substance useful for carrying out the composition of the Disclosure or the method of the Disclosure. Thus, the container may be a vial or an ampoule. It may be made from any suitable material, such as glass, plastic, metal, or paper or paper products. In embodiments, it may be a glass or plastic ampoule or vial, which may be sealed by a stopper, a stopper and crimp seal, or a plastic or metal cap. The amount of imaging agent contained in the container may be selected by a person skilled in the art without excessive experimentation, based on a number of relevant parameters according to the subject matter of the Disclosure.

[0063] In embodiments, a container is provided as a component of a larger unit, which typically includes packaging material (hereinafter referred to as a kit for simplicity). The kit of this disclosure may include instructions and / or other information relating to appropriate packaging and use of the composition. Typically, the kit may be made from a sturdy material, such as cardboard and plastic, and may include instructions or other information printed directly thereon. The kit may consist of a plurality of containers containing the composition of the present invention. In such a kit, each container may be the same size as each other and contain the same amount of composition, or different containers may be different sizes and / or contain different amounts of composition or compositions having different components. Those skilled in the art will immediately recognize that a number of different configurations of container sizes and contents are envisioned by the present invention, and therefore it is not necessary to specifically describe all permutations here.

[0064] Certain terminology is used here, but only in a generic and descriptive sense, and not for the purpose of limitation. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as they would be commonly understood by a person of ordinary skill in the art to which this subject now describes belongs.

[0065] In accordance with long-standing patent law convention, the terms “a” (indefinite article, a certain), “an” (indefinite article, used before a vowel), and “the” (definite article, the) refer to “one or more” (also called one or more) when used in this application, including in the claims. Thus, for example, a reference to “a certain subject” includes multiple subjects unless the context clearly indicates the opposite (e.g., multiple subjects).

[0066] Throughout this statement and claim, the terms “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense unless the context requires otherwise. Similarly, the term “include” and its grammatical variations are intended to be non-restrictive, so as not to exclude other similar items that may replace or add to the listed items from the recitation of items in the list.

[0067] For the purposes of this specification and the attached claims, unless otherwise indicated, all numbers used in this specification and the claims that represent quantities, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values ​​are understood in all cases to be modified by the term "approximately," even if the term "approximately" does not explicitly appear with its value, quantity, or range. Unless otherwise indicated, the numerical parameters described in the following specifications and attached claims may be approximate and / or greater or less as desired, depending on the desired properties to be obtained by the subject matter of this disclosure, but may reflect tolerances, conversion factors, rounding off, measurement errors and other similar factors, and other factors known to those skilled in the art. For example, when referring to values, the term “about” may mean that such variation from a specified amount is appropriate for performing the disclosed method or employing the disclosed composition, including variations of ±100% in some embodiments, ±50% in some embodiments, ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments.

[0068] Furthermore, when used in relation to one or more numbers or ranges of numbers, the term “about” should be understood to refer to all such numbers, including all numbers within the range, and to modify that range by extending boundaries above and below the stated numbers. A recitation of a range of numbers by endpoints includes all numbers contained within that range, for example, including their fractions, the entire integer, and encompasses that range (for example, a recitation from 1 to 5 includes 1, 2, 3, 4, and 5, as well as their fractions, for example, 1.5, 2.25, 3.75, 4.1, and other of the same kind) and any range within that range. [Examples]

[0069] The following examples are included to provide guidance to a person of ordinary skill in the art for practicing typical embodiments of the subject matter of this disclosure. In light of this disclosure and the general level of skill in the art, a person of skill can recognize that the following examples are intended to be illustrative only, and that a great many variations, modifications, and alterations can be adopted without deviating from the scope of the subject matter of this disclosure. The general description and specific examples that follow are intended for illustrative purposes only and should not be construed as limiting in any way the compounds of this disclosure can be made by any other means. Example 1 1.1 Results

[0070] 1.1.1 Synthesis and In Vitro Evaluation of Hydrophilic PD-L1 Specific PET Imaging Agents. PD-L1 detection using IHC is a guiding tool for PD-1:PD-L1 therapy. McLaughlin et al., 2016. However, tools for non-invasively quantifying total PD-L1 levels in all lesions have only recently emerged and are in the early stages of clinical evaluation. Bensch et al., 2018; Niemeyer et al., 2018. However, quantifying PD-L1 kinetics presents different challenges, given the need for PET imaging agents that provide high-contrast images within a standard clinical workflow.

[0071] To address this need, a PD-L1-specific peptide-based imaging agent is [ 64 Cu]WL12 was previously developed and its potential for detecting tumor PD-L1 levels was demonstrated. Kumar et al., 2019. However, [ 64 Cu]WL12 is lipophilic and shows high nonspecific accumulation in several tissues, including the liver. (Kumar et al., 2019; Chatterjee et al., 2017). To improve imaging properties, novel hydrophilic peptides were identified, and radiofluorinated analogs were generated using the aluminum fluoride method to facilitate clinical translation.

[0072] DK221 is a 14-amino acid human PD-L1-specific cyclic peptide containing three carboxylate groups and free lysineamine. Miller et al., 2016. The structure of DK221 is shown here. This is shown immediately below, and free lysineamine is annotated with an asterisk: [ka] DK221

[0073] To modify DK221 for radiolabeling, a bifunctional chelator (chelating agent), for example, NCS-MP-NODA was conjugated to free lysineamine to produce DK222. The NODA chelator is a non-radioactive analog [ 19 Similar to F]DK222, [ 18 [F]DK222 was used for radiofluorination (Figures 1A, 10, and 11). A competitive PD-1:PD-L1 suppression assay was performed to characterize the binding affinity of peptide analogs to PD-L1. The peptide analogs used were DK221, DK222, and [ 19F]DK222 ICs of 24, 28, and 25 nM respectively 50 The value was observed to suppress binding to PD-L1 in a dose-dependent manner (Figure 1B). Peptides and small molecules were analyzed by the aluminum fluoride (AlF) method. 18 Radiolabeled [F]fluoride is attracting attention due to its ease of synthesis and the possibility of retaining the hydrophilicity of the bound moisture. McBride et al., 2010; Kumar et al., 2018. Radiolabeled analogs, 18 [F]DK222 was synthesized by the AlF method, which exhibits good radiochemical yield (34.85±1.7%, n=62), in vitro stability, and a moderate specific activity of 284±56 mCi / μmol (10.51±2.07 GBq / μmol, n=25). [ 18 F]DK222 was found to be stable for 4 hours at the prepared radioactivity concentration (Figures 12, 13, and 14).

[0074] [ PD-L1 18 To evaluate the specificity of F]DK222, cell binding assays were performed. CHO cells with constitutive human PD-L1 expression (hPD-L1) and multiple cancer cell lines derived from triple-negative breast cancer (TNBC) (MDAMB231, SUM149) and melanoma (LOX-IMVI, MeWo, and A375) were selected. The cells were [ 18 The cells were incubated with F]DK222 at 4°C for 30 minutes, thoroughly washed, and their cell binding activity was measured. 18 F]DK222 import is F The variable levels of surface PD-L1 expression observed by low cytometry (Figure 1C and Figure 1D) were reflected in the order: hPD-L1 > LOX-IMVI > MDAMB231 > Sum149. A375, CHO, and MeWo cells expressed low PD-L1 levels and the least [ 18 It showed F]DK222 binding.

[0075] About PD-L1 [ 18To verify the specificity of [F]DK222, binding studies were performed in the presence of a 1 μM excess of the parent DK221 peptide. A greater than 90% reduction in radioactivity uptake was observed in PD-L1-positive cells (P<0.0001). In summary, these in vitro results suggest that [ 18 This provided evidence that the binding of F]DK222 is specific to PD-L1.

[0076] 1.1.2 In the mouse model of TNBC [ 18 Evaluation of the in vivo distribution of F]DK222.[ 18 To gain insights into the PK and in vivo distribution of F]DK222, we performed PET imaging studies in immunodeficient NSG mice carrying PD-L1-positive MDAMB231 xenografts. 18 PET images acquired at 15, 60, and 120 minutes after injection of [F]DK222 showed high accumulation of radioactive material in the tumor as early as 15 minutes (Figure 2A). In addition to the tumor, the kidney showed the highest uptake of radioactivity at all time points examined. [ 18 The high and selective uptake of F]DK222, combined with its rapid clearance from normal tissue, 18 High-contrast images were provided 60–120 minutes after injection of [F]DK222. Reduced uptake of the radiotracer was observed in SUM149 tumors expressing low PD-L1 levels and in mice receiving a blocking dose (50 mg / kg) of the parental DK221 peptide.

[0077] Ex vivo measurements at 5, 30, 60, 120, 240, and 360 minutes after radiotracer injection are used to validate imaging studies and in normal tissue. 18 This was performed to quantify the intracellular distribution of F]DK222 (Table 1). In NSG mice carrying MDAMB231 xenografts, [ 18 Ex vivo distribution study of F]DK222. [Table 1]

[0078] 18 The uptake of F]DK222 remained consistently high in tumors up to 4 hours after injection (Figure 2B). The time-activity curves plotted from the biodistribution data (Figure 2C) (shown as percentage of injected dose per gram of tissue [%ID / g]) showed high 18 accumulation and retention of F]DK222 in the MDA-MB-231 tumors. 18 A steady increase in the uptake of F]DK222 was observed in tumors up to 120 minutes, followed by a slow decline between 120 and 360 minutes. Consistent with the PET imaging, 18 the uptake of F]DK222 was consistently even higher in tumors and kidneys. Small peptides often demonstrate renal clearance, and the high uptake in the observed kidneys 18 indicates the renal clearance of F]DK222. It was observed that the radioactivity decreased steadily over time in blood, muscle, and all other tissues contributing to high image contrast. The tumor-to-blood and tumor-to-muscle ratios at 60 minutes were 4.5 ± 0.2 and 30.0 ± 1.3, respectively. High [[ID=​​​​​F]DK222 showed a 79% decrease (P<0.0001) in uptake. Furthermore, administration in the range of non-radioactive doses decreased uptake in a dose-dependent manner in MDAMB231 tumors, but not in SUM149 tumors or other tissues (Figures 15A - 15C). These observations were confirmed by the strong and weak immune responses to PD-L1 observed in MDAMB231 and SUM149 tumors, respectively (Figure 2D). Based on the observed high tumor uptake and high tumor-to-blood (and muscle) ratios, all imaging and biodistribution studies in other tumor models were performed here at 60 minutes as provided below.

[0079] 1.1.3. In melanoma xenograft models 18 In vivo verification of F]DK222 specificity. Next, the PD-L1 specificity of F]DK222 was verified in melanoma models. 18 When F]DK222 was injected into NSG mice bearing PD-L1 high-expressing LOX-IMVI or PD-L1 low-expressing MeWo melanoma xenografts, 18 F]DK222 showed high radioactivity accumulation in LOX-IMVI tumors. 18 The uptake of F]DK222 was low in MeWo tumors and in mice receiving blocking doses of non-radioactive peptides (Figures 3A and 3B, Figure 16). Consistent with the results of PET imaging, ex vivo measurement tests performed at 60 minutes showed that 18 the uptake of F]DK222 in MeWo tumors was 95% less compared to LOX-IMVI tumors (P<0.0001). Histological analysis supported the PET imaging findings, where strong PD-L1 immunoreactivity was observed in LOX-IMVI tumors (upper panel), but not in MeWo tumors (lower panel) (Figure 3C).

[0080] Similar results were also observed in lung and bladder cancer models (Figures 8 and 9). Overall, in vivo imaging and ex vivo measurements in TNBC and melanoma models [[ID=!9]] 18This provided further evidence regarding the specificity of F]DK222 and its potential to quantify variable PD-L1 levels across different tumor types.

[0081] 1.1.4.[ 18 Quantification of the pharmacodynamic effects of aPD-1 mAbs in tumors using [F]DK222. The PD-1 / PD-L1 pathway represents the cornerstone of combination immune checkpoint blockade regimens. Topalian et al., 2015. Many of these combination therapies converge on the production of IFNγ, which is closely linked to PD-L1 levels. Minn and Wherry, 2016. PD-L1 activation exhibits robust cytolytic activity that is suppressed by TME or released by PD-1-targeting therapeutic agents. Topalian et al., 2015; Minn and Wherry, 2016. Reinvigoration of depleted T cells can be detected in the blood as early as 3 weeks after receiving PD-1 therapeutic agents in patients. In contrast, its activity in tumors remains not fully understood. Huang et al., 2019. Immune reactivation in tumors is often accompanied by cytolytic activity of immune cells, IFNγ secretion, and induction of PD-L1 levels in the tumor bed. Taube et al., 2012. Thus, although we do not wish to be bound by any particular theory, tumor PD-L1 levels are a measure of the pharmacodynamic effect of PD-1 therapeutic substances. It was thought that this would be a proximal biomarker for this purpose. Therefore, it was necessary to quantify tumor PD-L1 levels induced by different PD-1 therapeutic agents using PD-L1 PET, and to confirm these measurements with immunological responses.

[0082] First, the effect of adaptive immune responses on PD-L1 expression in melanoma cells was investigated by evaluating changes in PD-L1 levels induced by IFNγ treatment. LOX-IMVI, A375, and MeWO melanoma cells treated with IFNγ were analyzed for changes in PD-L1 levels. Flow cytometry analysis showed that PD-L1 levels increased 2-fold and 4-fold, respectively, in response to IFNγ treatment in LOX-IMVI and A375 cells (Figure 17). No difference was observed in MeWo cells.

[0083] To quantify differences in tumor PD-L1 levels as a measure of adaptive immune response to treatment with different aPD-1 mAbs, a humanized mouse model was used. NSG mice humanized with PBMCs (huPBMCs) carrying A375 melanoma xenografts were treated with a single dose of aPD-1 mAbs (12 mg / kg). After one week of treatment, tumor PD-L1 levels were [ 18 Measured by [F]DK222-PET and ex vivo counting after 24 hours (Figure 4A). Controls included huPBMC mice with tumors treated with Serene, and NSG mice treated with pembrolizumab and nivolumab.

[0084] First, we evaluated whether there were differences in PD-L1 levels in tumors between humanized and non-humanized mice. All huPBMC mice had A375 tumors indicating immune cell activity. 18 This showed an increase in the uptake of F]DK222. On the other hand, in NSG mice that lack huPBMC, [ 18 F]DK222 uptake was low (%ID / g 8.5 vs 3.9; P=0.0002). Analysis of tumor sections showed increased immunoreactivity to PD-L1 and CD3 against NSG in huPBMC mice, and the results of PET testing were validated. [ 18 Increased uptake of F]DK222 was also observed compared to that of NSG mice (Figure 18). On the other hand, in nonspecific tissues, such as muscle, [18 No significant difference was observed in the uptake of F]DK222. These results are [ 18 F]DK222 indicated the potential to differentiate tumors with low PD-L1 levels, and possibly those that have been eliminated by immune cells.

[0085] Next, we evaluated the pharmacodynamic effects of different aPD-1 therapeutic substances in tumors. First, we examined the differences in tumor PD-L1 levels between treatment groups in huPBMC mice. Notably, 3 out of 6 huPBMC mice treated with the vehicle showed high levels. 18 This showed uptake of F]DK222. In contrast, a significant number of mice treated with aPD-1 mAbs showed high tumor development with some variability. 18 F]DK222 uptake was observed (Figures 4A and 4B). Validating PET imaging data and clarifying the differences in treatment-induced PD-L1 levels in TME, and studying in vivo distribution, compared to NSG mice, showed that mice treated with nivolumab, pembrolizumab, or Serene, respectively, were [ 18 F]DK222 uptake showed increases of 148%, 85%, and 76% in median values ​​(Figure 4C). Analysis of tumor sections from aPD-1 mAb-treated mice showed the observed [ 18 F]DK222 showed increased immunoreactivity for PD-L1 and CD3. These results indicate that different PD-1 therapeutic agents exert different PD effects in tumors, which can be measured as changes in tumor PD-L1 levels.

[0086] Observed [ 18 To verify that the uptake of F]DK222 is indeed PD-L1 specific, tumors were extracted from mice and flow cytometry analysis was performed to quantify PD-L1 levels. In the PD-1 treatment group, [ 18 Increased uptake of [F]DK222 and total PD-L1 levels were observed, supported by increased accumulation of CD45+CD8+ immune cells in the tumor (Figures 4D, 4E, and 4F). 18F]DK222 uptake and total PD-L1 levels in tumors (R 2 =0.80; P<0.0001, Figure 4G) and tumor cell-specific PD-L1 levels (R 2 A strong correlation was observed between the two (=0.71; P<0.0001). On the other hand, [ 18 F]DK222 uptake and immune cell-specific PD-L1 The correlation between levels is low, probably because the contribution of immune cell PD-L1 levels to the total PD-L1 levels in TME is small in this model (R 2 =0.57; P<0.0001). In addition, there is an increase in the accumulation of CD45+CD8+ cells in tumors in the treatment group. [ 18 The uptake of F]DK222 did not correlate with PD-L1 levels. These data are [ 18 F]DK222 This study demonstrates that PET can be used to quantify PD-L1 kinetics induced by aPD-1 treatment.

[0087] next,[ 18 To test the hypothesis that [F]DK222 uptake can be used to quantify the differential effects of aPD-1 mAbs in tumor sites, a fixed-effects model for statistical analysis was used to quantify heterogeneity in induced PD-L1 levels in tumor sites. In the fixed-effects model, both fixed aPD-1 mAbs are considered as specific choices compared to each other. We investigated which of nivolumab and pembrolizumab more effectively induced PD-L1 over time at a given dose. Each aPD-1 mAb was compared to Serene, and the differences are shown in a table. When the same dose was administered, a subtle, but not statistically significant, difference was observed between nivolumab and pembrolizumab in PD-L1 levels induced by nivolumab treatment, resulting in greater PD-L1 expression in this model system. In summary, these data were measured in tumor sites. 18We demonstrate that F]DK222 can be used to compare the pharmacodynamic effects of different aPD-1 mAbs early in the treatment process.

[0088] 1.1.5.a Quantification of accessible tumor PD-L1 levels during PD-L1 treatment. 18 To quantify the PD-L1 levels accessible using [F]DK222, the interaction of peptide analogs and aPD-L1 mAbs with the PD-L1 protein was first studied using biolayer interferometry. The dissociation constant of the PD-L1 peptide was found to be at least 100 times weaker than that of aPD-L1 mAbs. This observation was found at the tracer concentration used (low nM), [ 18 This suggests that F]DK222 does not interfere with anti-PD-L1 therapy. To replicate these observations in cell-based systems, MDAMB231 and LOX-IMVI cells were tested in the presence or absence of 60 nM PD-L1 mAb. 18 Incubated with [F]DK222 at 4°C for 30 minutes, and bound radioactivity was measured in both cell types in the presence of mAbs. 18 A greater than 65% decrease in F]DK222 uptake was observed (P<0.0001), indicating that cell membrane PD-L1 levels were occupied by the mAb (Figure 5A). These data [ 18 This indicates that F]DK222 has the potential to quantify PD-L1 levels accessible in vivo and can enable the quantification of PD-L1 levels accessible during processing.

[0089] To confirm in vitro observation in vivo, NSG mice with LOX-IMVI tumors were treated with a single dose of atezolizumab at 0.3 or 20 mg / kg, [ 18 F]DK222 was administered intravenously as a bolus 24 hours prior to infusion (Figures 5B, 5C, and 5D). 18PET images acquired 60 minutes after injection of [F]DK222 showed significant accumulation of radioactivity in tumors in vehicle-treated controls. In contrast, signal intensity in tumors was significantly reduced in mice treated with 20 mg / kg mAb (Figure 5C and Figure 5D). Importantly, there was a moderate reduction in signal intensity when using a low dose of 0.3 mg / kg atezolizumab. Ex vivo studies compared tumors in mice treated with 20 and 0.3 mg / kg atezolizumab, respectively, compared with vehicle-treated mice. 18 F]DK222 uptake showed 89% (P<0.0001) and 32% (P<0.01) decreases, indicating different accessible PD-L1 levels in tumors (Figure 5D, Figure 19). In summary, these in vitro and in vivo results suggest that [ ]DK222 uptake may be useful for measuring accessible tumor PD-L1 levels and for identifying lesions that are not saturated by drug treatment. 18 [F]DK222 demonstrates the potential of PET. 1.1.6. Accessible tumor PD-L1 levels provide insights into the PK and PD effects of aPD-L1 mAbs in the tumor site.

[0090] The efficacy of different mAbs targeting PD-L1 in TME may be heterogeneous due to the different PK and PD, and this remains unspecified. 18 F]DK222 can be coupled to an accessible PD-L1, and therefore, [ 18 The F]DK222 PET signal can indicate to some extent that PD-L1 remains inaccessible: a lower signal indicates better PD-L1 mAb targeting efficiency. Insights resulting from the mAb's PK and PD during the immunotherapy trial rounds could further guide the selection of specific mAbs for treatment.

[0091] The purpose of this experiment is to detect heterogeneity in the binding of different mAbs. 18The objective is to evaluate the potential of F]DK222, and for this purpose, to provide in principle that it can be used to guide the selection among several mAbs available for treatment. For this experiment, three mAbs were selected (atezolizumab, avelumab, durvalumab), Yu et al., 2019, and nivolumab was used as a priori negative control. Separate groups of animals were injected with a single 1 mg / kg dose of one of these mAbs, and after either 24 or 96 hours, each group was [ 18 Inject, image, or sacrifice with F]DK222, and [ 18 The F]DK222 signal was quantified (Figure 6A). At 24 hours between any PD-L1 mAb and nivolumab, an even lower [ 18 The difference in the F]DK222 signal constitutes the saturated coupling of PD-L1 mAb. On the other hand, at 96 hours, an even higher [ 18 The F]DK222 signal, compared to that after 24 hours, represents the loss of mAbs from its binding site, and more PD-L1 [ 18 Access to F]DK222 was made available. The experiment was repeated in two different tumor models: LOXIMVI and MDAMB231.

[0092] PET images of LOX-IMVI tumor-bearing mice were shown in all groups treated with aPD-L1 mAbs for 24 hours. 18 F]DK222 showed a significant decrease. On the other hand, in nivolumab-treated animals, [ 18 F]DK222 uptake was similar to that of vehicle-treated mice. In tumors of mice treated with atezolizumab and avelumab, at 96 hours [ 18 A significant increase in F]DK222 uptake was observed, but this was not the case in mice treated with durvalumab (Figure 6B). [ 18F]DK222 uptake was not significant, suggesting that uptake is specific to aPD-L1 mAb treatment. Further analyses were performed to verify these observations.

[0093] [ 18 To test the hypothesis that F]DK222 uptake can be used to quantify the differential effects of aPD-L1 mAbs, we used two different statistical analysis strategies to quantify the heterogeneity of therapeutic mAb binding. First, in a random-effects model, three selected mAbs were considered to be random samples of various available mAbs. This experiment was designed to answer the following question: "[ 18 The question is: "To what extent can the variance in the F]DK222 signal be explained by (a) the fact that different mAbs exist, or (b) that these mAbs may have different dynamics between 24 and 96 hours, rather than being grouped together as an active treatment?" To answer this question, three aPD-L1 mAbs were selected as random effects, and the overall difference in active treatment between PD-L1 mAbs and inactive mAbs, time points, and time points was selected as fixed effects. The random and fixed effects were jointly estimated in a mixed linear regression model, and heterogeneity was defined by the intraclass correlation coefficient (ICC).

[0094] ICC quantifies the proportion (or percentage) of total variance attributable to different active mAbs. ICC can range from 0 to 1 (from 0 to 100%), and a larger ICC indicates greater expected variance of PD and PK among various aPD-L1 mAbs. In a random PD-only effect model in LOX-IMVI tumors, the ICC was 0.23 (p-value = 8.9 × 10⁻⁶ vs. no random effect). -5 ) and [ 18It is indicated that 23% of the variance in the F]DK222 signal (%ID / g) arises from differences in PD-L1 occupancy. The ICC of the random PD-PK effects model is 0.36 (vs. p without random effects). Value = 3 × 10 -6 (vs. random PD model p-value = 0.0014), [ 18 It is indicated that 36% of the variance at %ID / g for F]DK222 stems from differences in PD and PK of different mAbs in the tumor environment. Similarly, in the MDAMB231 tumor model, the ICC for the sole effect model of random PD is 0.54 (p=0 vs. no random effect). The ICC for the random PD-PK effect model is 0.77 (p=0 vs. no random effect; p=0 vs. random PD model), indicating that 77% of the variance at %ID / g for MDAMB231 stems from differences in PD and PK of different mAbs in the tumor environment (Figure 20).

[0095] Secondly, in a fixed-effects model, each of the three fixed PD-L1 mAbs is considered a specific choice to be compared against the others. The question is: "Which of atezolizumab, avelumab, or durvalumab more effectively and specifically involves PD-L1 over time?" To answer this practical question, each PD-L1 mAb (specific saturated PD), each time point (overall PK, 24 and 96 hours), and each mAb*time combination (mAb-specific PK) were considered fixed effects and estimated together in a standard linear regression model. The results of this analysis are given as (a) the difference in accessible PD-L1 levels (%ID / g) for each PD-L1 mAbs versus nivolumab over 24 hours, and (b) for a specific mAb, the difference in accessible PD-L1 levels over 96 hours versus 24 hours (96h vs 24h) compared to the 96-to-24 hour difference for nivolumab.

[0096] In LOX-IMVI tumors and tissues of mice treated with different mAbs and time points, [ 18F]DK222 uptake is shown in Figure 6C. The mean LOX-IMVI tumor %ID / g at 24 hours was high in the nivolumab control (approximately 20%ID / g) and remained largely unchanged from 24 to 96 hours (Figure 6C). On the other hand, at 24 hours, all PD-L1 mAbs had significantly lower mean tumor %ID / g than nivolumab. Accessible PD-L1 levels at 96 hours were 60 to 80% higher than the levels observed at 24 hours of treatment in the atezolizumab and avelumab groups (P<0.001). However, accessible PD-L1 levels for durvalumab decreased by ≥70%, and were similar at 96 hours vs. 24 hours, suggesting that durvalumab is involved in PD-L1 long-term. These observations were validated in the MDAMB231 xenograft model (Figure 6D).

[0097] In short, random- and fixed-effects models reveal that PD-L1 mAbs have differential PK and PD in tumor sites, affecting accessible PD-L1 levels over time. Furthermore, these results clearly demonstrate the potential of PET to quantify accessible target levels and gain insights into the pharmacological activity of mAbs at tumor sites. 1.2 Discussion

[0098] mAbs conjugated with radionuclides are routinely used to gain insights into their in vivo distribution and target expression. Nearly 26 such drugs are in clinical trials. De Vries et al., 2019. Various mAbs, mAb-conjugates, and small proteins have been developed to detect PD-L1 expression. Josefsson et al., 2015; Maute et al., 2015; Chatterjee et al., 2016; Truillet et al., 2017; De Silva et al., 2018; Jagoda et al., 2019; Vento et al., 2019; Wissler et al., 2019; Hettich et al., 2016; Donnelly et al., 2018.

[0099] Recent studies using Zr-89-labeled atezolizumab have highlighted the potential of PET for quantifying intratumoral and intertumoral heterogeneity in PD-L1 expression. (Bensch et al., 2018). Despite these advances, there is a need for imaging agents that provide high-contrast images and fit into standard clinical workflows. Such high-contrast images are often observed with peptide and low molecular weight PET agents.

[0100] The subject matter of this disclosure is in part its [ 18This study demonstrates that the F]DK222 peptide exhibits distinct PK and in vivo distribution characteristics compared to reported PD-L1 imaging agents. (Bensch et al., 2018; Maute et al., 2015; Chatterjee et al., 2016; Truillet et al., 2017; De Silva et al., 2018; Jagoda et al., 2019; Donnelly et al., 2018; Lesniak et al., 2019; Heskamp et al., 2019; Ehlerding et al., 2019; Kikuchi et al., 2017; Chatterjee et al., 2017; Broos et al., 2017; Josefsson et al., 2016; Natarajan et al., 2015; Heskamp et al., 2015.)

[0101] Furthermore, now 18 F]DK222 possesses all the remarkable features necessary for routine clinical use: 1) high affinity and specificity for quantifying dynamic changes at the PD-L1 level; 2) easy-to-handle PK and low nonspecific accumulation in normal tissue compared to reported protein-based imaging agents, enabling its use across many tumor types; 3) adequate image contrast within 60 minutes of radiotracer administration, fitting within standard clinical workflows; and 4) human dosimetry estimates similar to other prominent PET imaging agents, such as those used for detecting prostate-specific membrane antigen and chemokine receptor 4. Szabo et al., 2015; Herrmann et al., 2015.

[0102] Accumulation of radiolabeled mAbs in tumors could indicate a tumor response to treatment. [In tumors acquired multiple days after radiotracer injection] 89The [Zr]atezolizumab signaling pathway was found to be a better predictor of tumor response to atezolizumab therapy than IHC and RNA sequencing-based predictive biomarkers. Bensch et al., 2018. However, in the ever-expanding PD-1 / PD-L1 therapeutic development arena, head-to-head comparisons between mAb therapeutic substances or to gain deeper insights into their differences in distribution and activity in tumors are needed. 89 ZrmAb imaging is impractical for clinical interpretation. Yu et al., 2019.

[0103] Here, we have radiopharmaceuticals with high affinity and rapid pharmacokinetics, for example, [ 18 Such as F]DK222 has been demonstrated to be useful beyond baseline PD-L1 level quantification and for treatment guidance. The potential of such assays for evaluating the in situ pharmacological activity of different aPD-L1 mAbs is demonstrated by the discovery of extended target engagement by durvalumab compared to other aPD-L1 mAbs in the preclinical models employed. Importantly, these PD assays encompass multiple factors influencing antibody concentration, including PD-L1 levels and turnover, the complex serum and tumor dynamics (or fate) of those mAbs in tumors, as well as tumor-endogenous parameters such as high interstitial pressure and poor vascularity that hinder antibody penetration and accumulation. Furthermore, these three mAbs have distinct PKs [atezolizumab (isotype IgG1κ, K] D , 0.4nM, t 1 / 2 Avelumab (IgG1λ, 0.7 nM, 6.1 days), and durvalumab (IgG1κ, 0.022 nM, 18 days) showed similar results, and while the tumor residence kinetics of these mAbs do not reflect the circulating half-life profile, they do reflect mAb affinity for PD-L1. (Tan et al., 2017)

[0104] The approach and findings of this study also have potential implications for improving treatment regimens and for drug development and evaluation. Predictive computational models are routinely used in the clinical development and administration of mAbs (Agoram, 2007; Agoram, 2009). However, personalized cancer treatment based on these mechanistic models can be biased due to the lack of translatability between preclinical information used and preclinical experiments and patients. The non-invasive measurements presented here were able to bridge that gap. Furthermore, various next-generation mAb therapeutic materials, such as probodies specifically activated at TME (Giesen et al., 2019), and those that facilitate simultaneous binding to multiple targets, are further enhanced. The emergence of multi-specific mAb conjugates, such as those described by Lan et al. (2018), which enable higher-avidity binding, may exhibit pharmacokinetics (PKs) different from those in conventional in silico models, and new approaches, such as measuring pharmacodynamic effects in tumors, will be necessary, taking into account their pharmacological activity in tumors.

[0105] It is important to note that DK222 is a more hydrophilic peptide and differs significantly in its in vivo distribution from other reported peptides, including WL12. WL12, being lipophilic, shows high liver, kidney, and nonspecific accumulation in several tissues. [Regarding MDAMB231 tumors...] 64 The tumor-to-blood and tumor-to-muscle ratios at 120 minutes after radiotracer infusion for Cu]WL12 were 12.9+2.1 and 2.72+0.45, respectively. 18 The tumor-to-blood and tumor-to-muscle ratios for F]DK222 were 35.69+3.89 and 9.45+0.51, respectively (specific activity: 250 mCi / μmole). Outside of tumors, [ 18High radioactivity uptake is observed only in the kidney, the organ involved in the clearance of F]DK222. This high tumor uptake and low background tissue uptake results in high-contrast PD-L1-specific images.

[0106] Furthermore, DK222 in this disclosure is also radiolabeled differently from previously reported peptide analogs (i.e., International PCT Patent Application Publication No. WO / 2017 / 201111 (PCT / US2017 / 033004), on PET-IMAGING IMMUNOMODULATORS, to Donnelly et al., published November 23, 2017, which is incorporated herein by reference in its entirety). The lysine ε-amine of DK221 was used for bifunctional chelator conjugation using the NHS ester method and required milder conditions than previously used methods, and it performed all conjugations at the aminoacetamide terminus, which required the incorporation of glycine during peptide synthesis or the use of harsh conditions for conjugation.

[0107] [ 18 The use of [F]AlF-based radiolabeling facilitates a one-step radiolabeling procedure that can be achieved within 60 minutes without the need for special equipment. The AlF radiolabeling strategy also preserves the hydrophilicity of the molecules required for the high-contrast images observed. While AlF radiolabeling has been previously reported with NOTA or NODAGA analogues, we observed even more robust and consistent radiofluorination with the described NODA analogue, further offering advantages over existing radiolabeling strategies applied for the development of PD-L1 imaging agents.

[0108] AlF is a Pyl radiolabeling strategy ([ 18Replacing it with [F]DK221-Py) alters the molecular PK and leads to poor image contrast, highlighting the importance of aluminum fluoride radiolabeled NODA conjugated DK221 for the development of optimal imaging agents. To achieve the desired contrast, some may add PEG or other linkers. 18 We were able to further regulate the in vivo pharmacokinetics and in vivo distribution of F]DK221-Py. 1.3 Materials and Methods

[0109] 1.3.1 Chemicals. DK221 has a purity of >95% and was custom synthesized by CPC Scientific (Sunnyvale, CA, USA). (2,2'-(7-(4-isothiocyanatobenzyl)-1,4,7-triazonan-1,4-diyl)diacetic acid) (NCS-MP-NODA) was purchased from Chematech Macrocycle Design Technologies (catalog number C110; Dijon, France). All other chemicals were purchased from Sigma-Aldrich or Fisher Scientific.

[0110] 1.3.2. Cell Culture Reagents and Antibodies. All cell culture reagents were purchased from Invitrogen (Grand Island, NY). aPD-L1 mAbs (atezolizumab, avelumab, durvalumab) and aPD-1 mAbs (nivolumab, pembrolizumab) were purchased from Johns Hopkins School of Medicine Pharmacy.

[0111] 1.3.3 Synthesis of DK222. DK221 is a 14-amino acid cyclic peptide having the sequence Cyclo(cyclic)-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-NMeNle-Lys-Cys-)-Gly-NH2. It was previously reported as peptide 6297. Miller et al., 2016. NODA conjugated analogue of DK221 (Cyclo-(-Ac-Tyr-NMeAla-Asn-Pro-His-Glu-Hyp-Trp-Ser-Trp(carboxymethyl)-NMeNle-Lys(NODA_NCS[ 18 [F]AlF)-Cys-)-Gly-NH2) was prepared as follows: In a 20 mL vial, DK221 (4.0 mg, 2.04 μmoles) was stirred in dimethylformamide (1.0 mL), to which diisopropylethylamine (5.0 μL) was added, followed by NCS-MP-NODA (1.6 mg, 4.07 μmoles). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified on a reverse-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 50-90% methanol (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 30 minutes. The desired DK222 was collected at 15.5 min, the solvent was evaporated, the residue was reconstituted in deionized water, and lyophilized to a powder with a 65% yield. The purified DK222 was characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Calculation [M+H] + :2348.68, Observation:2349.06 (Figures 10 and 11).

[0112] 1.3.4 [ 19Synthesis of DK222: A solution of 2 mL of NaF (1 M, 0.5 M NaOAc, pH 4) and 2 mL of AlCl3 (0.2 M, 0.5 M NaOAc, pH 4) was stirred in a 20 mL vial at room temperature for 10 minutes. To this vial, a prepared solution of DK222 (5 mg, 2.12 μmoles, 200 μL acetonitrile and 100 μL 0.5 M NaOAc, pH 4) was added and heated at 110 °C for 35 minutes. The reaction mixture was cooled to room temperature and evaporated to half of its total volume. The reaction mixture was loaded into a triple pre-activated Sep-Pak plus C18 cartridge and then washed with 5 mL of water (×5). 19 F]DK222 was eluted in 50% acetonitrile water (5 mL x 5). The collected fractions were combined, concentrated under rotavap, reconstituted in 20% acetonitrile water, and lyophilized to form an off-white powder in 80% yield. The resulting pure product was characterized by MALDI-TOF. Calculation [M=H] + :2392.65, observation:2393.03. Then, pure [ 19 The F]DK222 complex was used for optimizing RP-HPLC conditions, as a standard for radiolabeling, and for competitive binding assays of PD-L1 and PD-1. 19 The HPLC chromatogram and mass spectrometry of F]DK222 are shown in Figures 10 and 11. [ka]

[0113] 1.3.5. MALDI-TOF Analysis. MALDI-TOF spectra of DK222 and its precursors were obtained using a Voyager DE-STR MALDI-TOF available at the Johns Hopkins University Mass Spectrometry core facility. Briefly, the samples were equilibrated in water with 0.1% TFA using an Amicon Ultra-15 centrifugal filter unit (catalog UFC901008). The samples were mixed (1:2 dilution) with a 10 mg / ml sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) matrix dissolved in 40% acetonitrile and 0.1% TFA. 1 μL of these samples were spotted in a quadruplet onto MALDI plates (Applied Biosystems), air-dried, and spectra were acquired using optimal instrument settings. The data was analyzed using Applied Biosystems Data Explorer software version 4.8.

[0114] 1.3.6 [ 18 Preparation of radiopharmaceutical F]DK222. Received from the cyclotron at JHU PET Center. 18 [F] fluoride (without carrier) was captured on a pre-prepared Chromafix 30PS-HCO3 cartridge. The cartridge was then washed with metal-free water (5 mL). 18 F was eluted from the cartridge with 100 μL of 0.4 M KHCO3. The pH of the solution was adjusted to approximately 4 with 10 μL of metal-free glacial acetic acid, and then 2 mM AlCl was added in 0.1 M sodium acetate buffer (pH 4). 3· 20 μL of 6H2O was added. The resulting solution is Al 18The fluorine complex was incubated at room temperature for 2-4 minutes to form. The precursor DK222 (approximately 100 micrograms, 42 nmoles) was dissolved in 300 μL of a 2:1 solution of acetonitrile and NaOAc (0.1 M, pH 4), and then Al 18 It was added to a vial containing F. The resulting reaction mixture was heated at 110°C for 15 minutes. Next, the reaction vial was cooled to room temperature and diluted with 400 μL of DI Water (deionized water). The resulting aqueous solution containing the radiolabeled product was purified on an RP-HPLC system (Varian ProStar) equipped with an Agilent Technology 1260 Infinity photodiode array detector (Agilent Technologies, Wilmington, DE). A semi-preparative C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA) was used for gradient elution, starting with 50% methanol (0.1% TFA) and reaching 90% methanol at 30 minutes, with water (0.1% TFA) as a cosolvent at a flow rate of 5 mL / min. The radiolabeled product eluted with a retention time of approximately 16.2 minutes, [ 18 F]DK222 was collected, evaporated under high pressure, compounded with Serene containing 10% EtOH, sterile filtered, and used for in vitro and in vivo evaluation.

[0115] Radiochemical purity, chemical identity, and in vitro stability HPLC chromatograms are shown in Figures 11A-11C. [ka]

[0116] 1.3.7 Cell Culture. Seven cell lines were used for in vitro and in vivo evaluation: MDAMB231 and SUM149 (triple-negative breast cancer), LOX-IMVI, MeWo and A375 (melanoma), CHO, and CHO cells constitutively expressing PD-L1 (hPD-L1). MDAMB231, MeWo, A375, and CHO cells were purchased from the American Type Culture Collection and cultured as recommended. CHO cells constitutively expressing PD-L1 (hPD-L1) were generated in our laboratory and cultured as previously described. Chatterjee et al., 2016. The SUM149 cell line was obtained from Dr. Stephen Ethier, and the LOX-IMVI cell line was obtained from the NCI Developmental Therapeutic Program. All cell lines were validated by STR profiling at the Johns Hopkins Genetic Resources Facility. SUM149 cells were maintained in Ham's F-12 medium containing 5% FBS, 1% P / S, 5 μg / mL insulin, and 0.5 μg / mL hydrocortisone. All cell lines were cultured in the recommended medium in an incubator at 37°C with a 5% CO2 atmosphere. Human embryonic kidney (HEK) 293F cells (Thermo Life Technologies) used for protein expression were maintained in suspension in FreeStyle 293 expression medium (Thermo Life Technologies) containing 0.01% penicillin-streptomycin (Gibco) with 5% ambient CO2 at 37°C.

[0117] 1.3.8 Detection of PD-L1 expression by flow cytometry. 2 × 10⁶ in 100 μL of PBS. 5Individual cells were evaluated for PD-L1 surface expression by direct staining with Cy5-atezolizumab at 4°C for 30 minutes. Cy5-atezolizumab was prepared as previously described (Kumar et al., 2019). The cells were then washed and analyzed for mean fluorescence intensity (MFI) by flow cytometry. Adherent cells were detached using enzyme-free cell dissociation buffer (Thermo Fisher Scientific, Waltham, MA).

[0118] 1.3.9 [ 18 In vitro binding assay using F]DK222. hPD-L1 MDAMB231, MeWo, A375, Sum149, and CHO cells [ 18 In vitro binding of F]DK222 is 1 × 10 6 Cells are subjected to approximately 0.1 μCi in the presence or absence of 1 μM DK222 or 60 nM mAbs. 18 The measurement was performed by incubation with F]DK222 at 4°C for 30 minutes. After incubation, the cells were washed three times with ice-cold PBS containing 0.1% Tween 20 and counted using an automated gamma counter (1282 Compugamma CS, Pharmacia / LKB Nuclear, Inc., Gaithersburg, MD (Maryland)). 18 To demonstrate PD-L1-specific binding of [F]DK222, blocking was performed with 1 μM unmodified peptide DK221. All cellular radioactivity uptake studies were performed quadruple for each cell line and repeated three times.

[0119] 1.3.10. In Vivo Studies. All mouse studies were conducted through protocols approved by the Johns Hopkins University Animal Care and Use Committee (ACUC). Xenografts were established in five-to-six-week-old, male or female, non-obese, diabetic, severe combined immunodeficiency gamma (NSG) mice obtained from the Johns Hopkins University Immune Compromised Animal Core. huPBMC mice were purchased from the Jackson (JAX) Laboratory and used as-is for experiments.

[0120] 1.3.11 Xenotransplant model. Mouse is MDAMB231 (2×10 6 , orthotopic), SUM149 (5×10 6 (Isotopic), LOX-IMVI (5×10 6 , intradermal), MeWo (5×10 6 , intraderm), or A375 (2×10 6 Cells were implanted in the rostral end using intradermal cells. When two cell lines were used on the right side of the mouse, along with a cell line expressing high levels of PD-L1, cells were implanted on the opposite flank. Inoculated. Tumor volume is 200-400 mm 3 The mice were used for processing, imaging, or in vivo distribution experiments.

[0121] 1.3.12 PET-CT imaging of mouse xenografts. 18 To determine the in vivo distribution and pharmacokinetics of F]DK222, PET images were acquired at multiple time points. Mice with MDAMB231 tumors were given ~200 μCi (7.4 mBq) of [ 18F]DK222 was intravenously injected in 200 μL of Serene (n=3), and the animals were anesthetized under 3% isofluorane before being placed in the scanner. PET images were acquired at two bed positions at 5 minutes / bed, as described, at 15, 60, and 120 minutes after radiotracer injection, using an ARGUS small animal PET / CT scanner (Sedecal, Madrid, Spain). CT scans (512 projection) were performed at the end of each PET scan for anatomical co-registration. PET data were reconstructed using the two-dimensional ordered subsets-expectation maximization algorithm (2D-OSEM) and corrected for dead time and radioactive decay. %ID values ​​per cc were calculated based on a calibration factor derived from known radioactive quantities. Image fusion, visualization, and 3D rendering are done in Amira 6.1 (R) ( (R) This was achieved using the registered trademark designation Amira 6.1 (FEI, Hillsboro, OR (Oregon)) in the United States and other countries. PET or PET / CT images were acquired 60 minutes after radiotracer injection (one or two beds, 5 minutes / bed) in all other tumor models.

[0122] For all other PET imaging studies reported here, mice received approximately 200 μCi (7.4 mBq) in 200 μL of Celine. 18 F]DK222 was administered intravenously, and PET or PET / CT images were acquired at 5 minutes / bed 60 minutes after injection using an ARGUS small animal PET / CT scanner.

[0123] 1.3.13 Ex vivo in vivo distribution. To validate imaging studies, ex vivo in vivo distribution studies were conducted in mice carrying human tumor xenografts (MDAMB231, Sum149, LOX-IMVI, and MeWo) as described. Lesniak et al., 2016. Mice carrying MDAMB231 tumors were given 50 μCi (1.85 MBq) [ 18 F]DK222 was administered intravenously, and tissue was collected at 5, 30, 60, 120, 240, or 360 minutes post-injection. In all other tumor models, [ 18 A biodistribution study was performed 60 minutes after injection of F]DK222. For blocking tests, 2 mg / kg (50 μg) of unmodified peptide was injected simultaneously with a radioactive tracer. To facilitate radiation dose calculations, collected tissues included tumors, blood, thymus, heart, lungs, liver, stomach, pancreas, spleen, adrenal glands, kidneys, small and large intestines, ovaries, uterus, muscle, femur, brain, and bladder. The collected tissues were weighed and counted using an automated gamma counter (Perkin Elmer - 2480 Automatic Gamma counter - Wizard2 3" Wallac, Waltham, MA), and the percentage of injected dose per gram of tissue (%ID / g) was measured in a triple-lens system with signal attenuation correction and external [ 18 [F] Calculated based on normalization to the standard. The in vivo distribution data shown is the mean ± standard error of the mean (SEM).

[0124] For all other in vivo distribution studies reported here, mice received ~50 μCi (1.85 mBq) [ 18 F]DK222 was administered intravenously in 200 μL of Serene, and the in vivo distribution study was conducted. 18 The procedure was performed 60 minutes after infusion of F]DK222. Selected tissues (tumors, blood, heart, lungs, liver, spleen, kidneys, small intestine, and muscle) were collected, weighed, counted, and their %ID / g values ​​were calculated.

[0125] 1.3.14 aPD-1mAb drug administration study. huPBMC mice obtained from JAX Labs were given 2x10 6 A375 cells were subcutaneously transplanted at the rostral tip. Seven days after cell inoculation (average tumor volume = 80 ± 15 mm) 3 ), mice were randomized and treated with a single 12 mg / kg dose of nivolumab or pembrolizumab by intravenous injection (n=9 / group). huPBMC mice treated with Serene (n=4-5 / (group). NSG mice treated with nivolumab or pembrolizumab at a dose of 12 mg / kg were used as controls. Seven days after treatment, at least 3-5 mice per group were treated. 18 F]DK222 PET scans were obtained. Mice were used for in vivo distribution studies 8 days post-processing and 24 hours post-PET imaging, and the data were processed as described. Harvested tumors were cut in half and used for flow cytometry analysis or IHC analysis for PD-L1 and CD3.

[0126] 1.3.15 Flow Cytometry Analysis. After ex vivo in vivo distribution analysis, tumors and spleens were stored overnight at 4°C in MACS Tissue Storage Solution (Miltenyi Biotec #130-100-008). Tumors and spleens were dissociated the following day according to the manufacturer's instructions (Miltenyi Biotec 130-095-929). Briefly, each xenograft and spleen was cut into 3-4 mm pieces. For each tumor, the piece was suspended in 2.5 mL of RPMI-1640 medium containing 100 μL of enzyme H, 50 μL of enzyme R, and 12.5 μL of enzyme A. For each spleen, the piece was suspended in 2.5 mL of FACS buffer containing 50 μL of enzyme D and 15 μL of enzyme A. The recommended program for tumors and spleens is gentleMACS. TM Octo Dissociator with Heaters( TMThe sample was moved using a GentleMACS Octo Dissociator (Miltenyi Biotec #130-096-427) with a heater, which is a trademark in the United States and other countries. A short centrifugation step was performed to collect the sample material at the bottom of the tube. The sample was resuspended and passed through a strainer (70 μm for tumors, 30 μm for spleens), centrifuged at 300 x g for 7 minutes, the supernatant was discarded, and the cells were resuspended in 2.5 mL of FACS buffer. The cells were counted, and 1 x 10⁶ cells were obtained. 6 The cells were resuspended in 100 μL of Live / Dead aqua solution (ThermoFisher #L34965, reconstituted with 2 mL of PBS) in a 96-well plate. The cells were incubated for 15 minutes (in the dark, RT) and washed with 150 μL of PBS. Fc blocking was performed using Biolegend Tru Samples were treated with BioLegend True Stain (#422301 Fc Block (1 μL in 100 μL of FACS buffer)) and incubated for 10 minutes (in the dark, at 4°C). After washing with 150 μL of cold FACS buffer, the samples were stained with antibodies targeting markers of interest in 100 μL of FACS buffer at the following dilutions. [Table 2]

[0127] The samples were incubated for 15 minutes (in the dark, RT), washed with 200 μL of FACS buffer, and fixed with 200 μL of Fix / Perm (eBio Foxp3 staining kit # 00-5523-00: 1 vol Fix-Perm concentrate with 3 vols Diluent). The samples were resuspended the following day in 500 μL of FACS buffer in LSR II. All data were analyzed on FlowJo v10.4.1.

[0128] 1.3.16 Immunohistochemical Analysis. Immunohistochemical analysis of PD-L1 was performed on clone (E1L3N (R) )XP (R) The procedure was performed using rabbit anti-human PD-L1 (Cell Signaling, 13684, dilution: 1:250) as previously described. Gniadek et al., 2017. Immunohistochemical staining of CD3 was performed using Polyclonal Rabbit Anti-Human CD3 (Dako / Agilent, A0452, dilution: 1:100) from NDBio Inc. (Baltimore), using clones.

[0129] 1.3.17 aPD-L1 mAb drug administration study. To determine the effect of antibody doses on accessible PD-L1 levels in tumors, LOX-IMVI tumor-bearing mice were treated with a single intravenous bolus dose of atezolizumab (0.3 or 20 mg / kg), and mice 24 hours later were used for imaging (n=3-4) and in vivo distribution (n=6-8) studies.

[0130] To determine the temporal changes in accessible PD-L1 levels in tumors after treatment with PD-L1 therapeutic agents, LOX-IMVI tumor-bearing mice were treated with a single intravenous bolus dose of atezorzumab, avelumab, or durvalumab (1 mg / kg), and imaging (n=3) and in vivo distribution studies (n=8-18) were performed at 24 and 96 hours post-antibody treatment. The anti-PD-1 antibody nivolumab (1 mg / kg) and Serene were used as controls. Mice treated with therapeutic mAbs for 24 hours, or Serene as a control, were used, and 200 μCi in 200 μL of Serene was measured. 18 Intravenous injection with [F]DK222 was performed, and PET images were acquired one hour after radiotracer injection. Due to the large number of groups and mice involved, controls treated with Serene and nivolumab were included in every experiment, and data from multiple experiments were pooled. The study was repeated only by measuring the in vivo distribution in mice carrying MDAMB231 tumors.

[0131] 1.3.18 Data Analysis. Statistical analysis was performed using Prism 8 Software (GraphPad Software, La Jolla, CA). Unpaired Student's t-test and one- or two-way ANOVA were used for columnar, multi-column, and grouped analyses, respectively. Data represent mean ± SEM. A p-value < 0.05 was considered statistically significant. 1.4 Synthesis 1.4.1 DK221 equivalents 1.4.1.1 DK222 (DK221-NODA) [ka]

[0132] 1.4.1.1. Procedure: To a stirred solution of DK221 (4.0 mg, 2.04 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by NCS-MP-NODA (1.6 mg, 4.07 μmoles). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 50-90% methanol (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 30 minutes. The desired DK222 was collected at 15.5 min, the solvent was evaporated, the residue was reconstituted in deionized water, and lyophilized to a powder in 65% yield. The purified DK222 was characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Calculated [M+H]+: 2348.68, observed: 2349.06. The MALDI-TOF MS of DK222 is shown in Figure 21. 1.4.1.2 DK331 (DK221-Biotin) [ka]

[0133] 1.4.1.2. Procedure: To a stirred solution of DK221 (5.0 mg, 2.55 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by biotin-NHS-Ester (2.0 mg, 5.85 μmoles). The reaction mixture was stirred at room temperature for 3–4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20–60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK331 was lyophilized in powder form in 57% yield and characterized by ESI MS. Calculation [MH] - :2182.50, Observation:2181.1. ESI-MS of DK331 is shown in Figure 22. MALDI-MS of DK331 is shown in Figure 23. 1.4.1.3.DK225(DK221-NODAGA) [ka]

[0134] 1.4.1.3. Procedure: To a stirred solution of DK221 (4.0 mg, 2.04 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by NODAGA-NHS-Ester (2.0 mg, 2.73 μmoles). The reaction mixture was stirred at room temperature for 3–4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20–60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK225 was lyophilized in 62% yield and characterized by ESI MS. Calculation [M+H]+ : 2313.57; Observation: 2314.0. The ESI-MS of DK225 is shown in Figure 24. 1.4.1.4. DK223 (DK221-DOTA)

Chemical Structure

[0135] 1.4.1.4.A. Procedure: Diisopropylethylamine (5.0 μL) was added to a stirred solution of DK221 (5.0 mg, 2.55 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, followed by DOTA-NHS-Ester (2.0 mg, 2.62 μmoles). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified on a reverse-phase high-performance liquid chromatography (RP-HPLC) system using a semi-preparative C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 25 minutes at a flow rate of 5 mL / min of 20 - 60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid). The desired DK223 was lyophilized to a powder in 72% yield and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Calculated [M + H]+: 2342.62, Observation: 2343. The MALDI-MS of DK223 is shown in Figure 25. 1.4.1.5. DK385 (DK221-DOTAGA)

Chemical Structure

[0136] 1.4.1.5.A. Procedure: To a stirred solution of DK221 (5.0 mg, 2.55 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by DOTA-GA anhydrous (3.1 mg, 6.27 μmoles). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20-60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK385 was freeze-dried into powder form with a yield of 61%, and it was characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Calculated [M+H]+: 2400.65, observed: 2401.09. The MALDI-MS of DK385 is shown in Figure 26. 1.4.1.6.DK365(DK221-PEG12-NOTA) [ka]

[0137] 1.4.1.6.A. Step-1: Synthesis of DK254 1.4.1.6. Structure of Ai DK254 [ka]

[0138] 1.4.1.6.A.ii Procedure: To a stirred solution of DK221 (5.0 mg, 2.55 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by Fmoc-N-amide-dPE12-NHS ester (2.0 mg, 2.13 μmoles). The reaction mixture was stirred at room temperature for 3-4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20-60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK254 was freeze-dried into a powder with a yield of 53% and characterized by ESI MS. Calculation [M+Na+2H] 2+ Figure 27 shows the ESI MS of 1400.5 and 1400.4.DK254. 1.4.1.6.B. Step-2: Synthesis of DK265

[0139] 1.4.1.6. Structure of BiDK265 [ka]

[0140] 1.4.1.6.B.ii Procedure: A solution of DK254 (5 mg) in a 20 mL vial was stirred at room temperature for 2 hours in dimethylformamide:piperidine (1:1) (1.0 mL). The crude reaction mixture was evaporated to dryness and used in the next step without further purification. Crude DK265 was obtained quantitatively and characterized by ESI MS. Calculation [M+2H] 2+ Figure 28 shows the ESI-MS of :1277.7, observation:1277.5.DK265. 1.4.1.6.C. Step-3: Synthesis of DK365

[0141] 1.4.1.6.Ci Procedure: To a stirred solution of DK265 (5.0 mg, 2.0 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by pSCN-Bn-NOTA (2.0 mg, 3.57 μmoles). The reaction mixture was stirred at room temperature for 3–4 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20–60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK365 was lyophilized in 45% yield and characterized by ESI MS. Calculation [M+2H] 2+ :1502.2, Observation:1502.4. The ESI-MS of DK365 is shown in Figure 29. 1.4.1.7.DK360(DK221-PEG12-NODA) [ka]

[0142] 1.4.1.7.i. Procedure: To a stirred solution of DK265 (5.0 mg, 2.0 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, diisopropylethylamine (5.0 μL) was added, followed by NCS-MP-NODA (1.2 mg, 3.0 μmoles). The reaction mixture was stirred at room temperature for 2-3 hours. The reaction mixture was purified on a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a semi-prepared C-18 Luna column (5 mm, 10 × 250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 20-60% acetonitrile (0.1% trifluoroacetic acid) and H2O (0.1% trifluoroacetic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK360 was lyophilized in 47% yield and characterized by ESI MS. Calculation [M+2H] 2+: 1473.0, Observation: 1472.7. The ESI-MS of DK360 is shown in Figure 30. 1.4.1.8. DK388 (DK221-PEG4-alkyne) [Chemical Structure]

[0143] 1.4.1.8.i. Procedure: Diisopropylethylamine (5.0 μL) was added to a stirred solution of DK265 (5.0 mg, 2.0 μmoles) in dimethylformamide (1.0 mL) in a 20 mL vial, followed by NCS-MP-NODA (1.2 mg, 3.0 μmoles). The reaction mixture was stirred at room temperature for 2 - 3 hours. The reaction mixture was purified on a reverse-phase high performance liquid chromatography (RP-HPLC) system using a semi-preparative C-18 Luna column (5 mm, 10×250 mm Phenomenex, Torrance, CA). The HPLC conditions for purification were 30 - 60% acetonitrile (0.1% formic acid) and H2O (0.1% formic acid) at a flow rate of 5 mL / min for 25 minutes. The desired DK388 was lyophilized to a powder in 58% yield and was characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Calculated [M] + : 2198.48, Observation: 2198.91. The ESI-MS of DK388 is shown in Figure 31. 1.4.1.9. 18 F] Synthesis of DK221Py [Chemical Structure]

[0144] Crude 18 F] The RP-HPLC of PyTFP is shown in Figure 32. Crude 18 F] The RP-HPLC of DK221Py is shown in Figure 33. Pure 18 F] The RP-HPLC of pure DK221Py is shown in Figure

[0145] Furthermore, in hPD-L1 / CHO xenografts [ 18 The in vivo evaluation of F]DK221Py is shown in Figure 35. References

[0146] All publications, patent applications, patents, and other references referenced in this specification indicate the level of expertise of a person in the art relating to the subject matter of this disclosure. All publications, patent applications, patents, and other references referenced in this specification (e.g., websites, databases, etc.) are incorporated herein by reference in whole to the same extent as each individual publication, patent application, patent, and other reference is specifically and individually indicated as being incorporated by reference. While some patent applications, patents, and other references are referenced herein, it will be understood that such references do not constitute an acknowledgment that any of these documents form part of the common general knowledge in the art. In the event of any inconsistency between this specification and the incorporated references, this specification (including any modifications thereof, which may be based on the incorporated references) shall prevail. Here, unless otherwise indicated, the standard technically accepted meaning of terms is used. Here, standard abbreviations for various terms are used.

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[0148] The aforementioned subject matter has been explained in some detail with illustrations and examples for the purpose of clarifying understanding, but it will be understood by those skilled in the art that certain changes and modifications can be put into practice within the scope of the attached claims.

Claims

1. Compound of formula (I): 【Chemistry 1】 An imaging agent containing [this ingredient].

2. 94mTc, 166 , 82 , 166 , 18 , 89 , 152 Tc, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 186 Re, 188 Re, 60 [[ID=Z]]Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 55 Co, 57 Co, <00R0017>Sc, 47 Sc, 225 Ac, <00000RR>Bi, 212 Bi, 212 Pb, 153 Sm, 166 Ho, 152 Gd, 82 Rb, 89 Zr, 166 Dy, and Al 18 The imaging agent according to claim 1, further comprising a radioactive metal selected from the group consisting of F. It should be noted that there may be some inaccuracies in the original text and the translation due to the unclear or incorrect tags in the original. You may need to check and correct the relevant content for a more accurate translation.

3. In detecting programmed death ligand 1 (PD-L1), the following is performed: (a) To provide an effective amount of the imaging agent of claim 1 or 2; (b) bringing one or more cells or tissues into contact with the imaging agent; and (c) Creating an image to detect PD-L1 This includes imaging methods, but excludes methods for imaging humans.

4. The imaging method of claim 3, wherein contact between one or more cells or tissues and the imaging agent is performed in vitro, in vivo, or ex vivo.

5. The imaging method of claim 4, wherein contact between one or more cells or tissues and the imaging agent is performed in a non-human subject.

6. The imaging method of claim 5, wherein the subject is a rat, mouse, cat, dog, horse, sheep, cattle, monkey, bird, or amphibian.

7. The imaging method of claim 3, wherein PD-L1 detection is performed approximately 60–120 minutes or less after administration of an imaging agent to a non-human subject.

8. The imaging method of claim 3, wherein the imaging method is used to detect cancer.

9. The imaging method of claim 8, wherein the cancer is selected from the group consisting of blastoma, calcinoma, glioma, leukemia, lymphoma, melanoma, myeloma, sarcoma, head cancer, cervical cancer, head and neck cancer, lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia / lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary tract cancer, pancreatic cancer, digestive tract cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, brain cancer, adenoma, and metastatic cancer.

10. The imaging method of claim 3, wherein the imaging method is used to detect a solid tumor.

11. The imaging method of claim 10, wherein the solid tumor is located in an organ selected from the group consisting of the brain, colon, breast, prostate, liver, kidney, lung, esophagus, head and neck, ovary, cervix, stomach, rectum, bladder, uterus, testes, and pancreas.

12. The imaging method of claim 3, wherein the imaging method is used to detect infection.

13. The imaging method of claim 12, wherein the infection is a microbial infection.

14. Microbial infections include Mycobacterium tuberculosis, Escherichia coli, Klebsiella sp., Enterobacter sp., Proteus sp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus spp., including Staphylococcus aureus and coagulase-negative Staphylococcus, Enterococcus sp., Streptococcus pneumoniae, and Haemophilus influenzae. The imaging method of claim 13, comprising infection by one or more microorganisms selected from the group consisting of influenzae, Bacteroides spp., Acinetobacter spp., Helicobacter spp., Candida sp., methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VRE).

15. The imaging method of claim 3, wherein the imaging method is used to detect inflammation.

16. The imaging method of claim 15, wherein the inflammation is associated with a disorder selected from the group consisting of asthma, autoimmune disease, autoinflammatory disease, celiac disease, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivity, inflammatory bowel disease, interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, lupus, systemic lupus erythematosus, and vasculitis.

17. The imaging method of claim 16, wherein the inflammation is caused by rheumatoid arthritis or systemic lupus erythematosus.

18. The imaging method of claim 3, wherein the imaging method is used to detect one or more immune cells in a tumor.

19. The imaging method of claim 3, wherein the imaging method is used to detect the systemic distribution of immune cells in a tumor or in a non-human subject.

20. The imaging method of claim 3, wherein the imaging method is used to detect the response of immune cells to an infectious disease.

21. The imaging method of claim 3, wherein the imaging method is used to detect the response of immune cells in a tumor or in normal tissue to an inflammatory disease.

22. The imaging method according to claim 3, wherein the imaging method detects the PD-L1 expression level in a non-human subject.

23. The imaging method according to claim 3, wherein the imaging method measures the occupancy of PD-L1 in a tumor site or normal tissue of a non-human subject.

24. A kit for detecting programmed death ligand 1 (PD-L1), comprising an imaging agent according to claim 1 or 2.

Images