Small-animal PET Imaging Studies Research Articles

Clinical translation of a novel FAPI dimer [68Ga]Ga-LNC1013.

Three new FAPI dimers were synthesized by linking two quinoline-based FAPIs with different spacers. The in vitro binding affinity and preclinical small animal PET imaging of the compounds were compared with their monomeric counterparts, FAPI-04 and FAPI-46. The lead compound, [68Ga]Ga -LNC1013, was then evaluated in a pilot clinical PET imaging study involving seven patients with gastrointestinal cancer. The three newly synthesized FAPI homodimers had high binding affinity and specificity in vitro and in vivo. Small animal PET imaging and biodistribution studies showed that [68Ga]Ga-LNC1013 had persistent tumor retention for at least 4h, also higher uptake than the other two dimers and the monomer counterparts, making it the lead compound to enter clinical investigation. In the pilot clinical PET imaging study, seven patients were enrolled. The effective dose of [68Ga]Ga-LNC1013 was 8.24E-03mSv/MBq. The human biodistribution of [68Ga]Ga-LNC1013 demonstrated prominent tumor uptake and good tumor-to-background contrast. [68Ga]Ga-LNC1013 PET imaging showed potential in capturing primary and metastatic lesions and outperforming 18F-FDG PET in detecting pancreatic and esophageal cancers. The SUVmax for lesions with [68Ga]Ga-FAPI-46 decreased over time, whereas [68Ga]Ga-LNC1013 exhibited persistently high tumor uptake from 1 to 4h post-injection. Dimerization is an effective strategy to produce FAPI derivatives with favorable tumor uptake, long tumor retention, and imaging contrast over its monomeric counterpart. We demonstrated that [68Ga]Ga-LNC1013, the lead compound without any piperazine moiety, had superior diagnostic potential over [68Ga]Ga-FAPI-46 and 18F-FDG, suggesting the future potential of LNC1013 for radioligand therapy of FAP-positive cancers.

Abstract A016: Novel positron emission tomography imaging targeting cell surface glycans for pancreatic cancer: 18F-labeled rBC2LCN lectin

Abstract Introduction: Advancement in early detection modalities will greatly improve the overall prognoses of pancreatic ductal adenocarcinoma (PDAC). As a molecular imaging technique, [18F]FDG-PET imaging has provided new insight in PDAC diagnosis; however, the utility of FDG for early-stage PDAC detection remains undefined. Therefore, PET radiotracers conjugated to malignancy-specific targeting agents (e.g., antibodies targeting cancer-associated/specific antigens) have been developed to improve specificity toward cancer cells but persist in the developmental stage. One underexplored cancer cell surface-specific target is the glycocalyx. We have previously demonstrated that fucosylated glycans (H type-3 antigens) are expressed in pancreatic cancers and a recombinant lectin, rBC2LCN, binds to these glycans specifically (Shimomura O et al: Mol Cancer Ther 2018). Here, we developed a novel, lectin-based PET probe targeting cell surface glycan for pancreatic cancer. Material and Methods: The rBC2LCN (16kDa) lectin was labeled with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), prepared according to literature (Maeding P, et al: Appl Radiat Isot 2005) in phosphate buffer (pH 8.5). Obtained [18F]FB-rBC2LCN was then purified using gel filtration column (PD-10, GE, Uppsala, Sweden). A PDAC cell line, Capan-1, was used as a model which expresses H type-3 antigens. The biodistribution of the [18F]FB-rBC2LCN was evaluated in athymic nude mice bearing subcutaneous Capan-1 tumors. Static, small-animal PET imaging studies (G4, SOFIE, Dulles, VA) were performed to further evaluate in vivo tumor targeting and specificity. Results: The decay-corrected radiochemical mean yield of [18F]FB-rBC2LCN was 3.6%±0.3% (mean±SD), based on [18F]SFB, and radiochemical purity was above 95%. Cell binding and uptake revealed that [18F]FB-rBC2LCN binds to Capan-1 cells. Mice received 0.32±0.16 MBq (corresponding to 59±21 mg) of product, injected intravenously. Biodistribution studies demonstrated that Capan-1 tumor uptake (6.5±2.0 %ID/g) was high as early as 60 min after injection of [18F]FB-rBC2LCN and the uptake increased over time (9.6±2.2 and 11±3.9 at 150 and 240 min after injection, respectively). Tumor-to-muscle ratio increased with time up to 19±1.8 at 360 min after injection, indicating image contrast. Although kidneys (72±23 %ID/g), liver (7.1±1.7 %ID/g) and lungs (7.4±2.9 %ID/g) showed high uptake at 60 min after injection, the uptake of those organs decreased over time. The blood activity concentration of the tracer was also high at 60 min after injection (12±4.4 %ID/g) and decreased over time. Bone uptake was very low (below 1.8 %ID/g throughout the study), indicating that defluorinating metabolism was negligible. High-contrast PET imaging of tumors was achieved as early as 60 min after injection and the contrast became clearer over 240 min, consistent with the biodistribution result. Conclusions: Our 18F-labeled rBC2LCN lectin warrants further clinical development to improve the accuracy and sensitivity of early-stage PDAC detection. Citation Format: Yukihito Kuroda, Tatsuya Oda, Osamu Shimomura, Bryan J. Mathis, Hiroaki Tateno, Kentaro Hatano. Novel positron emission tomography imaging targeting cell surface glycans for pancreatic cancer: 18F-labeled rBC2LCN lectin [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A016.

Immuno-PET of colorectal cancer with a CEA-targeted [68Ga]Ga-nanobody: from bench to bedside.

An accurate diagnosis of colorectal carcinoma (CRC) can assist physicians in developing reasonable therapeutic regimens, thereby significantly improving the patient's prognosis. Carcinoembryonic antigen (CEA)-targeted PET imaging shows great potential for this purpose. Despite showing remarkable abilities to detect primary and metastatic CRC, previously reported CEA-specific antibody radiotracers or pretargeted imaging are not suitable for clinical use due to poor pharmacokinetics and complicated imaging procedures. In contrast, radiolabeled nanobodies exhibit ideal characteristics for PET imaging, for instance, rapid clearance rates and excellent distribution profiles, allowing same-day imaging with sufficient contrast. In this study, we developed a novel CEA-targeted nanobody radiotracer, [68Ga]Ga-HNI01, and assessed its tumor imaging ability and biodistribution profile in preclinical xenografts and patients with primary and metastatic CRC. The novel nanobody HNI01 was acquired by immunizing the llama with CEA proteins. [68Ga]Ga-HNI01 was synthesized by site-specifically conjugating [68Ga]Ga with tris(hydroxypyridinone) (THP). Small-animal PET imaging and biodistribution studies were performed in CEA-overexpressed LS174T and CEA-low-expressed HT-29 tumor models. Following successful preclinical assessment, a phase I study was conducted on 9 patients with primary and metastatic CRC. Study participants received 151.21 ± 25.25MBq of intravenous [68Ga]Ga-HNI01 and underwent PET/CT scans at 1h and 2h post injection. Patients 01-03 also underwent whole-body dynamic PET imaging within 0-40min p.i. All patients underwent [18F]F-FDG PET/CT imaging within 1week after [68Ga]Ga-HNI01 imaging. Tracer distribution, pharmacokinetics, and radiation dosimetry were calculated. [68Ga]Ga-HNI01 was successfully synthesized within 10min under mild conditions, and the radiochemical purity was more than 98% without purification. Micro-PET imaging with [68Ga]Ga-HNI01 revealed clear visualization of LS174T tumors, while signals from HT-29 tumors were significantly lower. Biodistribution studies indicated that uptake of [68Ga]Ga-HNI01 in LS174T and HT-29 was 8.83 ± 3.02%ID/g and 1.81 ± 0.87%ID/g, respectively, at 2h p.i. No adverse events occurred in all clinical participants after the injection of [68Ga]Ga-HNI01. A fast blood clearance and low background uptake were observed, and CRC lesions could be visualized with high contrast as early as 30min after injection. [68Ga]Ga-HNI01 PET could clearly detect metastatic lesions in the liver, lung, and pancreas and showed superior ability in detecting small metastases. A significant accumulation of radioactivity was observed in the kidney, and normal tissues physiologically expressing CEA receptors showed slight uptakes of [68Ga]Ga-HNI01. An interesting finding was that strong uptake of [68Ga]Ga-HNI01 was found in non-malignant colorectal tissues adjacent to the primary tumor in some patients, suggesting abnormal CEA expression in these healthy tissues. [68Ga]Ga-HNI01 is a novel CEA-targeted PET imaging radiotracer with excellent pharmacokinetics and favorable dosimetry profiles. [68Ga]Ga-HNI01 PET is an effective and convenient imaging tool for detecting CRC lesions, particularly for identifying small metastases. Furthermore, its high specificity for CEA in vivo makes it an ideal tool for selecting patients for anti-CEA therapy.

Dynamic Tumor-Specific MHC-II Immuno-PET Predicts the Efficacy of Checkpoint Inhibitor Immunotherapy in Melanoma.

Despite the advance of immunotherapy, only a small subset of patients gains long-term survival benefit. This fact represents a compelling rationale to develop immuno-PET imaging that can predict tumor response to immunotherapy. An increasing number of studies have shown that tumor-specific major histocompatibility complex II (tsMHC-II) is associated with improved responses to targeted immunotherapy. The aim of this study was to investigate the potential of tsMHC-II protein expression and its dynamic change on treatment with interferon γ (IFNγ) as a new target for immuno-PET to predict response to immunotherapy. Methods: Major histocompatibility complex II (MHC-II) antibody was radiolabeled with DOTA-chelated 64Cu to derive an MHC-II immuno-PET tracer. Two melanoma models (B16SIY, B16F10) that are respondent and nonrespondent, respectively, to PD1/PD-L1 checkpoint inhibitor were used. Both tumor models were treated with anti-PD1 and IFNγ, enabling observation of dynamic changes in tsMHC-II. Small-animal PET imaging, biodistribution, and histologic studies were performed to validate the correlation of tsMHC-II with the tumor response to the immunotherapy. Results: Fluorescence-activated cell sorting analysis of the 2 tumors supported the consensual recognition of tsMHC-II correlated with the tumor response to the immunotherapy. The invivo PET imaging revealed higher basal levels of tsMHC-II in the responder, B16SIY, than in the nonresponder, B16F10. When treated with anti-PD1 antibody in animals, B16SIY tumors displayed a sensitive increase in tsMHC-II compared with B16F10 tumors. In IFNγ stimulation groups, the greater magnitude of tsMHC-II was further amplified when the IFNγ signaling was activated in the B16SIY tumors, as IFNγ signaling positively upregulates tsMHC-II in the tumor immunity. Subsequent histopathologic analysis supported the correlative characteristics of tsMHC-II with tumor immunity and response to cancer immunotherapy. Conclusion: Collectively, the predictive value of tsMHC-II immuno-PET was validated for stratifying tumor immunotherapy responders versus nonresponders. Monitoring sensitivity of tsMHC-II to IFNγ stimulation may provide an effective strategy to predict the tumor response to immunotherapy.

A new class of PentixaFor- and PentixaTher-based theranostic agents with enhanced CXCR4-targeting efficiency.

Non-invasive PET imaging of CXCR4 expression in cancer and inflammation as well as CXCR4-targeted radioligand therapy (RLT) have recently found their way into clinical research by the development of the theranostic agents [68Ga]PentixaFor (cyclo(D-Tyr1-D-[NMe]Orn2(AMBS-[68Ga]DOTA)-Arg3-Nal4-Gly5) = [68Ga]DOTA-AMBS-CPCR4) and [177Lu/90Y]PentixaTher (cyclo(D-3-iodo-Tyr1-D-[NMe]Orn2(AMBS-[177Lu/90Y]DOTA)-Arg3-Nal4-Gly5) = [177Lu/90Y]DOTA-AMBS-iodoCPCR4). Although convincing clinical results have already been obtained with both agents, this study was designed to further investigate the required structural elements for improved ligand-receptor interaction for both peptide cores (CPCR4 and iodoCPCR4). To this aim, a series of DOTA-conjugated CPCR4- and iodoCPCR4-based ligands with new linker structures, replacing the AMBA-linker in PentixaFor and PentixaTher, were synthesized and evaluated.Methods: The in vitro investigation of the novel compounds alongside with the reference peptides PentixaFor and PentixaTher encompassed the determination of hCXCR4 and mCXCR4 affinity (IC50) of the respective natGa-, natLu-, natY- and natBi-complexes in Jurkat and Eμ-myc 1080 cells using [125I]FC-131 and [125I]CPCR4.3 as radioligands, respectively, as well as the evaluation of the internalization and externalization kinetics of selected 68Ga- and 177Lu-labeled compounds in hCXCR4-transfected Chem-1 cells. Comparative small animal PET imaging studies (1h p.i.) as well as in vivo biodistribution studies (1, 6 and 48h p.i.) were performed in Daudi (human B cell lymphoma) xenograft bearing CB17 SCID mice.Results: Based on the affinity data and cellular uptake studies, [68Ga/177Lu]DOTA-r-a-ABA-CPCR4 and [68Ga/177Lu]DOTA-r-a-ABA-iodoCPCR4 (with r-a-ABA = D-Arg-D-Ala-4-aminobenzoyl-) were selected for further evaluation. Both analogs show app. 10-fold enhanced hCXCR4 affinity compared to the respective references [68Ga]PentixaFor and [177Lu]PentixaTher, four times higher cellular uptake in hCXCR4 expressing cells and improved cellular retention. Unfortunately, the improved in vitro binding and uptake characteristics of [68Ga]DOTA-r-a-ABA-CPCR4 and -iodoCPCR4 could not be recapitulated in initial PET imaging studies; both compounds showed similar uptake in the Daudi xenografts as [68Ga]PentixaFor, alongside with higher background accumulation, especially in the kidneys. However, the subsequent biodistribution studies performed for the corresponding 177Lu-labeled analogs revealed a clear superiority of [177Lu]DOTA-r-a-ABA-CPCR4 and [177Lu]DOTA-r-a-ABA-iodoCPCR4 over [177Lu]PentixaTher with respect to tumor uptake (18.3±3.7 and 17.2±2.0 %iD/g, respectively, at 1h p.i. vs 12.4±3.7%iD/g for [177Lu]PentixaTher) as well as activity retention in tumor up to 48h. Especially for [177Lu]DOTA-r-a-ABA-CPCR4 with its low background accumulation, tumor/organ ratios at 48h were 2- to 4-fold higher than those obtained for [177Lu]PentixaTher (except for kidney).Conclusions: The in-depth evaluation of a series of novel CPCR4- and iodoCPCR4 analogs with modified linker structure has yielded reliable structure-activity relationships. It was generally observed that a) AMBA-by-ABA-substitution leads to enhanced ligand internalization, b) the extension of the ABA-linker by two additional amino acids (DOTA-Xaa2-Xaa1-ABA-) provides sufficient linker length to minimize the interaction of the [M3+]DOTA-chelate with the receptor, and that c) introduction of a cationic side chain (Xaa2) greatly enhances receptor affinity of the constructs, obliterating the necessity for Tyr1-iodination of the pentapeptide core to maintain high receptor affinity (such as in [177Lu]PentixaTher).As a result, [177Lu]DOTA-r-a-ABA-CPCR4 has emerged from this study as a powerful second-generation therapeutic CXCR4 ligand with greatly improved targeting efficiency and tumor retention and will be further evaluated in preclinical and clinical CXCR4-targeted dosimetry and RLT studies.

Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention.

Cancer-associated fibroblasts constitute a vital subpopulation of the tumor stroma and are present in more than 90% of epithelial carcinomas. The overexpression of the serine protease fibroblast activation protein (FAP) allows a selective targeting of a variety of tumors by inhibitor-based radiopharmaceuticals (FAPIs). Of these compounds, FAPI-04 has been recently introduced as a theranostic radiotracer and demonstrated high uptake into different FAP-positive tumors in cancer patients. To enable the delivery of higher doses, thereby improving the outcome of a therapeutic application, several FAPI variants were designed to further increase tumor uptake and retention of these tracers. Methods: Novel quinoline-based radiotracers were synthesized by organic chemistry and evaluated in radioligand binding assays using FAP-expressing HT-1080 cells. Depending on their in vitro performance, small-animal PET imaging and biodistribution studies were performed on HT-1080-FAP tumor–bearing mice. The most promising compounds were used for clinical PET imaging in 8 cancer patients. Results: Compared with FAPI-04, 11 of 15 FAPI derivatives showed improved FAP binding in vitro. Of these, 7 compounds demonstrated increased tumor uptake in tumor-bearing mice. Moreover, tumor–to–normal-organ ratios were improved for most of the compounds, resulting in images with higher contrast. Notably two of the radiotracers, FAPI-21 and -46, displayed substantially improved ratios of tumor to blood, liver, muscle, and intestinal uptake. A first diagnostic application in cancer patients revealed high intratumoral uptake of both radiotracers already 10 min after administration but a higher uptake in oral mucosa, salivary glands, and thyroid for FAPI-21. Conclusion: Chemical modification of the FAPI framework enabled enhanced FAP binding and improved pharmacokinetics in most of the derivatives, resulting in high-contrast images. Moreover, higher doses of radioactivity can be delivered while minimizing damage to healthy tissue, which may improve therapeutic outcome.

Synthesis and Preclinical Characterization of the PSMA-Targeted Hybrid Tracer PSMA-I&F for Nuclear and Fluorescence Imaging of Prostate Cancer.

The prostate-specific membrane antigen (PSMA)–targeted radiotracers 68Ga/177Lu-PSMA-I&T and 99mTc-PSMA-I&S (for imaging and surgery) are currently successfully used for clinical PET imaging, radionuclide therapy, and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a PSMA-I&T–based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated. Methods: The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T, and their corresponding natGa-/68Ga- and natLu/177Lu counterparts was determined in LNCaP cells via competitive binding assays (IC50) and dual-tracer radioligand and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17 SCID and LNCaP xenograft–bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of 1 tumor-bearing animal as well as PSMA immunohistochemistry and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed. Results: Compared with the parent PSMA-I&T analogs, the PSMA affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9 < IC50 < 10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94%–95%), and very low accumulation in nontarget organs was observed already at 1 h after injection. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5% ± 1.8 percentage injected dose per gram) and the kidneys (106% ± 23 percentage injected dose per gram). Tumor-to-background ratios of 2.1, 5.2, 9.6, and 9.6 for blood, liver, intestines, and muscle, respectively, at 1 h after injection led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by immunohistochemistry. Conclusion: Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T–based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

Synthesis and in vitro and in vivo evaluation of urea-based PSMA inhibitors with increased lipophilicity

BackgroundSeveral radiolabeled prostate-specific membrane antigen (PSMA) inhibitors based on the lysine-urea-glutamate (KuE) motif as the pharmacophore proved to be suitable tools for PET/SPECT imaging of the PSMA expression in prostate cancer patients. PSMA I&T, a theranostic tracer developed in our group, was optimized through alteration of the peptidic structure in order to increase the affinity to PSMA and internalization in PSMA-expressing tumor cells. However, further structural modifications held promise to improve the pharmacokinetic profile.ResultsAmong the investigated compounds 1–9, the PSMA inhibitors 5 and 6 showed the highest PSMA affinity (lowest IC50 values) after the introduction of a naphthylalanine modification. The affinity was up to three times higher compared to the reference PSMA I&T. Extended aromatic systems such as the biphenylalanine residue in 4 impaired the interaction with the lipophilic binding pocket of PSMA, resulting in a tenfold lower affinity. The IC50 of DOTAGA-conjugated 10 was slightly increased compared to the acetylated analog; however, efficient PSMA-mediated internalization and 80% plasma protein binding of 68Ga-10 resulted in effective tumor targeting and low uptake in non-target tissues of LNCaP tumor-bearing CD-1 nu/nu mice at 1 h p.i., as determined by small-animal PET imaging and biodistribution studies. For prolonged tumor retention, the plasma protein binding was increased by insertion of 4-iodo-d-phenylalanine resulting in 97% plasma protein binding and 16.1 ± 2.5% ID/g tumor uptake of 177Lu-11 at 24 h p.i.ConclusionsHigher lipophilicity of the novel PSMA ligands 10 and 11 proved to be beneficial in terms of affinity and internalization and resulted in higher tumor uptake compared to the parent compound. Additional combination with para-iodo-phenylalanine in the spacer of ligand 11 elevated the plasma protein binding and enabled sustained tumor accumulation over 24 h, increasing the tumor uptake almost fourfold compared to 177Lu-PSMA I&T. However, high renal uptake remains a drawback and further studies are necessary to elucidate the responsible mechanism behind it.

Development of 64Cu-NOTA-Trastuzumab for HER2 Targeting: A Radiopharmaceutical with Improved Pharmacokinetics for Human Studies.

The purpose of this study was to develop 64Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2 (HER2). Methods: Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with 64Cu. Serum stability and immunoreactivity of 64Cu-NOTA-trastuzumab were tested. Small-animal PET imaging and biodistribution studies were performed in a HER2-positive breast cancer xenograft model (BT-474). The internal dosimetry for experimental animals was determined using the image-based approach with the Monte Carlo N-particle code. Results:64Cu-NOTA-trastuzumab was prepared with high radiolabeling yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of 64Cu-NOTA-trastuzumab was highest at 48 h after injection as determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations of 64Cu-NOTA-trastuzumab decreased biexponentially with time in both mice with and mice without BT-474 tumor xenografts. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 mGy/MBq for the liver, and 0.047 mGy/MBq for the spleen. Conclusion:64Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor in vitro and in vivo, and it exhibited a relatively low absorbed dose due to a short residence time. Therefore, 64Cu-NOTA-trastuzumab could be applied to select the right patients and right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.

A novel molecular agent for glioma angiogenesis imaging.

Gliomas are rich in blood vessels and are the most primary and malignant type of brain tumor affecting the central nervous system. A few fluorine-18 (F)-labeled imaging agents can be used for imaging of tumor angiogenesis. In the current study, F-labeled recombinant human endostatin (rh-endostatin) was developed and evaluated as a probe for PET imaging of tumor angiogenesis. F-fluorobenzoyl-endostatin (F-FB-endostatin) was synthesized from radiolabeling of rh-endostatin with N-succinimidyl-4-F-fluorobenzoate produced by a facile module-assisted radiosynthesis procedure. Blocking studies were used to measure the relative affinities of F-FB-endostatin to human glioblastoma U87MG cells in tumor tissues rich with vessels. In addition, biodistribution, metabolic stability, and small-animal PET imaging studies were carried out with F-FB-endostatin using Institute of Cancer Research and U87MG tumor-bearing mice. Noninvasive small-animal PET imaging indicated that F-FB-endostatin showed rapid and good tumor uptake. The probe was rapidly cleared from the blood and most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Metabolite assays showed that the probe was highly stable, making it suitable for in-vivo applications. F-FB-endostatin shows promising in-vivo properties. Therefore, the promising properties of F-FB-endostatin indicate that this probe can be a powerful tool to evaluate the antiangiogenic therapy for gliomas and thus warrants further investigation as a novel PET probe for imaging of tumor angiogenesis.

64Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor-Expressing Tumors.

The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 64Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-N,N',N,″-triacetic acid trihydrochloride ([p-SCN-Bn]-NOTA), 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-N-hydroxysuccinimide ester), or 1-(p-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([p-SCN-Bn]-DTPA) in 1.0 M NaHCO3 buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with 64Cu in 0.1 M NH4OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non-small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor-bearing mice. Results: Radiochemical yields of the 64Cu-labeled repebodies were approximately 70%-80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor-bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). Conclusion: The 3 64Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET.

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display.

Purpose: Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients.Experimental design: For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed in vitro using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified in situ by using peptide histochemistry, in vitro using flow cytometry, and in vivo by positron emissions tomography (PET/CT).Results: We identified a novel ITGαvβ6 binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity (KD = 14.8 nmol/L) for ITGαvβ6 In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of 68Ga- and 177Lu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions.Conclusions: Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαvβ6-positive carcinoma. Clin Cancer Res; 23(15); 4170-80. ©2017 AACR.

Surface charge and particle size determine the metabolic fate of dendritic polyglycerols.

Dendritic polyglycerols (dPG) are water soluble, polyether-based nanomaterials which hold great potential in diagnostic as well as therapeutic applications. In order to translate them for in vivo applications, a systematic assessment regarding their cell and tissue interactions as well as their metabolic fate in vivo is a crucial step. Herein, we explore the structure-activity relationship of three different sizes (ca. 3, 5, and 10 nm) of neutral dendritic polyglycerol (dPG) and their corresponding negatively charged sulfate analogs (dPGS) on their in vitro and in vivo characteristics. Cellular metabolic activity was studied in A431 and HEK293 cells. Biomolecular corona formation was determined using an electrophoretic mobility shift assay, which showed an increased protein binding of the dPGS even with serum concentrations as low as 20%. An in situ technique, microscale thermophoresis, was employed to address the binding affinities of these nanomaterials with serum proteins such as serum albumin, apo-transferrin, and fibrinogen. In addition, nanoparticle-cell interactions were studied in differentiated THP-1 cells which showed a charge dependent scavenger receptor-mediated uptake. In line with this data, detailed biodistribution and small animal PET imaging studies in Wistar rats using 68Ga-labeled dPG-/dPGS-NOTA conjugates showed that the neutral dPG-NOTA conjugates were quantitatively excreted via the kidneys with a subsequent hepatobiliary excretion with an increase in their size, whereas the polysulfated analogs (dPGS-NOTA) were sequestered preferentially in the liver and kidneys irrespective of their size. Taken together, this systematic study accentuates that the pharmacokinetics of dPGs is critically dependent on the overall size and charge and can be, fine-tuned for the intended requirements in nano-theranostics.

Novel Bispecific PSMA/GRPr Targeting Radioligands with Optimized Pharmacokinetics for Improved PET Imaging of Prostate Cancer

A new series of bispecific radioligands (BRLs) targeting prostate-specific membrane antigen (PSMA) and gastrin releasing peptide receptor (GRPr), both expressed on prostate cancer cells, was developed. Their design was based on the bombesin (BN) analogue, H2N-PEG2-[D-Tyr(6),β-Ala(11),Thi(13),Nle(14)]BN(6-14), which binds to GRPr with high affinity and specificity, and the peptidomimetic urea-based pseudoirreversible inhibitor of PSMA, Glu-ureido-Lys. The two pharmacophores were coupled through copper(I)-catalyzed azide-alkyne cycloaddition to the bis(tetrafluorophenyl) ester of the chelating agent HBED-CC via amino acid linkers made of positively charged His (H) and negatively charged Glu (E): -(HE)n- (n = 0-3). The BRLs were labeled with (68)Ga, and their preliminary pharmacological properties were evaluated in vitro (competitive and time kinetic binding assays) on prostate cancer (PC-3, LNCaP) and rat pancreatic (AR42J) cell lines and in vivo by biodistribution and small animal PET imaging studies in both normal and tumor-bearing mice. The IC50/Ki values determined for all BRLs essentially matched those of the respective monomers. The maximal cellular uptake of the BLRs was observed between 20 and 30 min. The BRLs showed a synergistic ability in vivo by targeting both PSMA (LNCaP) and GRPr (PC-3) positive tumors, whereas the charged -(HE)n- (n = 1-3) linkers significantly reduced the kidney and spleen uptake. The bispecific (PSMA and GRPr) targeting ability and optimized pharmacokinetics of the compounds developed in this study could lead to their future application in clinical practice as more sensitive radiotracers for noninvasive imaging of prostate cancer (PCa) by PET/CT and PET/MRI.

Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer.

Despite many advances in the past years, the treatment of metastatic prostate cancer still remains challenging. In recent years, prostate-specific membrane antigen (PSMA) inhibitors were intensively studied to develop low-molecular-weight ligands for imaging prostate cancer lesions by PET or SPECT. However, the endoradiotherapeutic use of these compounds requires optimization with regard to the radionuclide-chelating agent and the linker moiety between chelator and pharmacophore, which influence the overall pharmacokinetic properties of the resulting radioligand. In an effort to realize both detection and optimal treatment of prostate cancer, a tailor-made novel naphthyl-containing DOTA-conjugated PSMA inhibitor has been developed. The peptidomimetic structure was synthesized by solid-phase peptide chemistry and characterized using reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Subsequent (67/68)Ga and (177)Lu labeling resulted in radiochemical yields of greater than 97% or greater than 99%, respectively. Competitive binding and internalization experiments were performed using the PSMA-positive LNCaP cell line. The in vivo biodistribution and dynamic small-animal PET imaging studies were investigated in BALB/c nu/nu mice bearing LNCaP xenografts. The chemically modified PSMA inhibitor PSMA-617 demonstrated high radiolytic stability for at least 72 h. A high inhibition potency (equilibrium dissociation constant [K(i)] = 2.34 ± 2.94 nM on LNCaP; K(i) = 0.37 ± 0.21 nM enzymatically determined) and highly efficient internalization into LNCaP cells were demonstrated. The small-animal PET measurements showed high tumor-to-background contrasts as early as 1 h after injection. Organ distribution revealed specific uptake in LNCaP tumors and in the kidneys 1 h after injection. With regard to therapeutic use, the compound exhibited a rapid clearance from the kidneys from 113.3 ± 24.4 at 1 h to 2.13 ± 1.36 percentage injected dose per gram at 24 h. The favorable pharmacokinetics of the molecule led to tumor-to-background ratios of 1,058 (tumor to blood) and 529 (tumor to muscle), respectively, 24 h after injection. The tailor-made DOTA-conjugated PSMA inhibitor PSMA-617 presented here is sustainably refined and advanced with respect to its tumor-targeting and pharmacokinetic properties by systematic chemical modification of the linker region. Therefore, this radiotracer is suitable for a first-in-human theranostic application and may help to improve the clinical management of prostate cancer in the future.

Synthesis and radiopharmacological evaluation of 64Cu-labeled bombesin analogs featuring a bis(2-pyridylmethyl)-1,4,7-triazacyclononane chelator

The bifunctional chelating agent 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid, DMPTACN-COOH, has been found to bind strongly to copper(II), resulting in a radiocopper(II)-ligand complex that exhibits high in vivo stability. The pendant carboxylic acid group enables this derivative to be conjugated to the N-terminal amino acid residues of peptides. Exploiting this, two stabilized bombesin (BBN) derivatives, βAla-βAla-[Cha13,Nle14]BBN(7–14) and βhomo-Glu-βAla-βAla-[Cha13,Nle14]BBN(7–14) have been coupled to DMPTACN-COOH and radiolabeled with the positron emitter copper-64 (64Cu-1 and 64Cu-3). The in vitro binding characteristics of the [64Cu]Cu-labeled bombesin conjugates in gastrin-releasing peptide receptor (GRPR) over-expressing prostate cancer (PC-3) cells have been evaluated. Biodistribution studies performed in Wistar rats indicate a specific uptake in the GRPR-rich pancreas and rapid renal elimination for both 64Cu-1 and 64Cu-3. Small animal PET imaging studies performed in NMRI nu/nu mice bearing the human prostate tumor PC-3 demonstrated a very high degree of tumor accumulation for 64Cu-1 and 64Cu-3. Incorporation of a single additional glutamic acid residue within the spacer between bombesin and the radiolabeled complex (64Cu-3) leads to a higher tumor-to-muscle uptake ratio (amounting to >30 at 100 min post injection) compared to 64Cu-1.

Development and validation of an immuno-PET tracer for patient stratification and therapy monitoring of antibody-drug conjugate therapy.

11006 Background: SAR566658 is an antibody-drug immunoconjugate consisting of a humanized monoclonal antibody (huDS6) against the tumor-associated MUC1-sialoglycotope, CA6, conjugated to a cytotoxic maytansinoid (DM4). SAR566658 is currently undergoing phase I clinical trials in patients with CA6-positive solid tumors. A companion diagnostic based on huDS6 may facilitate patient stratification and early evaluation of therapeutic efficacy. The present study describes the development and preclinical evaluation of three novel Copper-64 (t½= 12.7h) labeled antibody fragments (two Fabs and a diabody) derived from the huDS6 antibody. One fragment was chosen based on imaging figures of merit for further specificity evaluations. Methods: The affinity of all fragments and their DOTA conjugates to CA6 was validated using flow cytometry. The DOTA conjugates were labeled with Copper-64 and evaluated in human serum stability studies, in vivo small animal PET imaging and 24-hour biodistribution studies in nude mice bearing either CA6 positive (WISH) or CA6 negative (A2780) subcutaneous tumors. The specificity of the lead tracer was evaluated in vivo via blocking studies and isotype controls. Results: All fragments and their DOTA conjugates had high affinity (Kd = 4-20 nM) for WISH cells. 64Cu-DOTA-B-Fab gave superior results in radio-synthesis (RCY - 60%, SA - 55 GBq/µmole, &gt;99% purity), serum stability (94±5%, n=3) and biodistribution profile. Its two isotype controls gave statistically significant (P&lt;0.05) uptake values in WISH but not A2780 tumors (see table). Blocking with B-Fab or huDS6 prior to tracer administration afforded a 23% (p&lt;0.05, n=8) and 26% (p&lt;0.05, n=7) decrease in WISH tumor uptake, respectively. Conclusions: These preclinical studies suggest that 64Cu-DOTA-B-Fab may be a suitable companion diagnostic for SAR566658 in cancer patients and requires further investigation.[Table: see text]

A simplified synthesis of the hypoxia imaging agent 2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-[(18)F]pentafluoropropyl)-acetamide ([18F]EF5).

[(18)F]EF5 is a validated marker for PET imaging of tumor hypoxia. It is prepared by reacting a trifluoroallyl precursor with carrier-added [(18)F]F(2) gas in trifluoroacetic acid (TFA) solvent. We report here an improved radiosynthesis and purification of [(18)F]EF5 by utilizing an electroformed nickel (Ni) target for [(18)F]F(2) production, and Oasis® HLB cartridges for on-line solid phase extraction of [(18)F]EF5 prior to HPLC purification. [(18)F]F(2) was produced by deuteron bombardment of neon plus F(2) in an Ni target, and bubbled through the radiolabelling precursor solution. Purification was achieved by extracting the contents of the crude reaction mixture onto Oasis HLB cartridges, and subsequently eluted onto a semi-preparative HPLC column for further separation. Purified [(18)F]EF5 was evaluated in small animal PET studies using HCT116 tumor xenografts in nude mice. The electroformed Ni target enabled recovery of >75% of the radioactivity from the cyclotron target, resulting in 16.2 ± 2.2 GBq (438 ± 58 mCi) of [(18)F]F(2) available for the synthesis. Use of Oasis cartridges yielded a less complex mixture for purification. On average, 1140 ± 200 MBq (30.8 ± 5.4 mCi) of [(18)F]EF5 were collected at EOS. Small animal PET imaging studies showed specific retention of [(18)F]EF5 in tumors, with tumor-to-muscle ratios of 2.7 ± 0.3 at about 160 min after injection. A simple procedure has been developed for the routine synthesis of [(18)F]EF5 in amounts and purity required for clinical studies. This new method avoids the need for TFA evaporation and also enables facile automation of the synthesis using commercially available radiosynthesis modules.

Synthesis and comparative biological evaluation of l- and d-isomers of 18F-labeled fluoroalkyl phenylalanine derivatives as tumor imaging agents

L-amino acid-based tracers have established their important role as tumor metabolic imaging agents. Recently, a number of studies demonstrated that D-amino acids may have improved imaging properties than their corresponding L-isomers. We synthesized and evaluated the D-isomer of a new phenylalanine derivative, p-(2-[(18)F]fluoroethyl)-phenylalanine ([(18)F]FEP), in comparison to its L-isomer and previously reported the L- and D-isomers of O-(2-[(18)F]fluoroethyl)-tyrosine ([(18)F]FET). L- and D-Isomers of [(18)F]FET and [(18)F]FEP were successfully synthesized via a rapid and efficient two-step nucleophilic fluorination and deprotection reaction. In vitro studies were carried out in 9L glioma cells. In in vivo studies, Fisher 344 rats bearing the 9L tumor model were used. L- and D-Isomers of (18)F-fluoroalkyl tyrosine and phenylalanine derivatives were efficiently labeled with high enantiomeric purity (>95%), good yield (11-45%) and high specific activity (21-75 GBq/μmol). D-[(18)F]FEP showed a similar linear time-dependent uptake as D-[(18)F]FET, while their corresponding L-isomers had much faster and higher uptake (4.3- to 16.0-fold at maximum uptake). The maximum uptake of the new compounds, L- and D-[(18)F]FEP, was 1.4- and 5.2-fold of that reported for L- and D-[(18)F]FET, respectively. Transport characterization studies indicated that both L- and D-[(18)F]FEP were selective substrates for system L. While L-[(18)F]FEP exhibited preference towards one subtype of system L, LAT1, D-[(18)F]FEP did not exhibit the same preference. Small animal PET imaging studies showed that both L- and D-[(18)F]FEP had higher uptake in 9L tumor compared to surrounding tissues, but D-isomer had lower tumor-to-muscle ratio in comparison with its L-isomer. Both L- and D-[(18)F]FEP are substrates for system L amino acid transporter with different preference toward its subtypes. Small animal imaging studies of 9L tumor showed that D-[(18)F]FEP did not show better imaging properties than their corresponding L-isomer.

5-[18F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression

The preliminary in vivo evaluation of novel 5-[(18)F]fluoroalkyl-2'-deoxyuridines ([(18)F]FPrDU, [(18)F]FBuDU, [(18)F]FPeDU; [(18)F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[(18)F]fluoroalkyl-1-beta-d-arabinofuranosyl uracils ([(18)F]FFPrAU, [(18)F]FFBuAU, [(18)F]FFPeAU; [(18)F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. [(18)F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [(18)F]F(-). For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). Biodistribution studies at 2 h pi revealed that the uptake of [(18)F]1a-b and [(18)F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [(18)F]1c and [(18)F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [(18)F]1c and [(18)F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [(18)F]1c and [(18)F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. Biological evaluations suggest that [(18)F]1c and [(18)F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.