Lingyi Sun

Novel Strategy for Preparing Dual-Modality Optical/PET Imaging Probes via Photo-Click Chemistry

Preparation of small molecule based dual-modality probes remains a challenging task due to the complicated synthetic procedure. In this study, a novel concise and generic strategy for preparing dual-modality optical/PET imaging probes via photo-click chemistry was developed, in which the diazole photo-click linker functioned not only as a bridge between the targeting-ligand and the PET imaging moiety, but also as the fluorophore for optical imaging. A dual-modality AE105 peptidic probe was successfully generated via this strategy and subsequently applied in the fluorescent staining of U87MG cells and the (68)Ga based PET imaging of mice bearing U87MG xenograft. In addition, dual-modality monoclonal antibody cetuximab has also been generated via this strategy and labeled with (64)Cu for PET imaging studies, broadening the application of this strategy to include the preparation of macromolecule based imaging probes.

Preclinical immunoPET/CT imaging using Zr-89-labeled anti-PD-L1 monoclonal antibody for assessing radiation-induced PD-L1 upregulation in head and neck cancer and melanoma

ABSTRACT Radiation therapy (RT) can induce upregulation of programmed death ligand 1 (PD-L1) on tumor cells or myeloid cells, which may affect response to PD-1-based immunotherapy. PD-L1 upregulation during RT is a dynamic process that has been difficult to monitor during treatment. The aim of this study was to evaluate the RT-induced PD-L1 upregulation in the tumor and its microenvironment using immunoPET/CT imaging of two syngeneic murine tumor models (HPV+ head and neck squamous cell carcinoma (HNSCC) or B16F10 melanoma). Tumors were established in two locations per mouse (neck and flank), and fractionated RT (2 Gy × 4 or 2 Gy × 10) was delivered only to the neck tumor, alone or during anti-PD-1 mAb immunotherapy. PD-L1 expression was measured by PET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and results were validated by flow cytometry. PET/CT imaging demonstrated significantly increased tracer uptake in irradiated neck tumors compared with non-irradiated flank tumors. Ex vivo analysis by biodistribution and flow cytometry validated PD-L1 upregulation specifically in irradiated tumors. In the HNSCC model, RT-induced PD-L1 upregulation was only observed after 2 Gy × 10 fractionated RT, while in the B16F10 model upregulation of PD-L1 occurred after 2 Gy × 4 fractionated RT. Fractionated RT, but not anti-PD-1 therapy, upregulated PD-L1 expression on tumor and infiltrating inflammatory cells in murine models, which could be non-invasively monitored by immunoPET/CT imaging using Zr-89 labeled anti-mouse PD-L1 mAb, and differentially identified anti-PD-1 responsive as well as selectively irradiated tumors in vivo.

New Bifunctional Chelator p-SCN-PhPr-NE3TA for Copper-64: Synthesis, Peptidomimetic Conjugation, Radiolabeling, and Evaluation for PET Imaging

Bifunctional chelators play an important role in developing metallic radionuclide-based radiopharmaceuticals. In this study, a new bifunctional ligand, p-SCN-PhPr-NE3TA, was synthesized and conjugated to a very late antigen-4 targeting peptidomimetic, LLP2A, for evaluating its application in (64)Cu-based positron emission tomography (PET) imaging. The new ligand exhibited strong selective coordination of Cu(II), leading to a robust Cu complex, even in the presence of 10-fold Fe(III). The LLP2A conjugate of p-SCN-PhPr-NE3TA was prepared and successfully labeled with (64)Cu under mild conditions. The conjugate (64)Cu-NE3TA-PEG4-LLP2A showed significantly higher specific activity, compared with (64)Cu-NOTA-PEG4-LLP2A, while maintaining comparable serum stability. Subsequent biodistribution studies and PET imaging in mice bearing B16F10 xenografts confirmed its favorable in vivo performance and high tumor uptake with low background, rendering p-SCN-PhPr-NE3TA a promising bifunctional chelator for (64)Cu-based radiopharmaceuticals.

Universal Molecular Scaffold for Facile Construction of Multivalent and Multimodal Imaging Probes.

Multivalent and multimodal imaging probes are rapidly emerging as powerful chemical tools for visualizing various biochemical processes. Herein, we described a bifunctional chelator (BFC)-based scaffold that can be used to construct such promising probes concisely. Compared to other reported similar scaffolds, this new BFC scaffold demonstrated two major advantages: (1) significantly simplified synthesis due to the use of this new BFC that can serve as chelator and linker simultaneously; (2) highly efficient synthesis rendered by using either click chemistry and/or total solid-phase synthesis. In addition, the versatile utility of this molecular scaffold has been demonstrated by constructing several multivalent/multimodal imaging probes labeled with various radioisotopes, and the resulting radiotracers demonstrated substantially improved in vivo performance compared to the two individual monomeric counterparts.

SW43-DOX ± loading onto drug-eluting bead, a potential new targeted drug delivery platform for systemic and locoregional cancer treatment – An in vitro evaluation

Treatment of unresectable primary cancer and their distant metastases, with the liver representing one of the most frequent location, is still plagued by insufficient treatment success and poor survival rates. The Sigma‐2 receptor is preferentially expressed on many tumor cells making it an appealing target for therapy. Thus, we developed a potential targeted drug conjugate consisting of the Sigma‐2 receptor ligand SW43 and Doxorubicin (SW43‐DOX) for systemic cancer therapy and for locoregional treatment of primary and secondary liver malignancies when loaded onto drug‐eluting bead (DEB) which was compared in vitro to the treatment with Doxorubicin alone.

Targeted Yttrium 89-Doxorubicin Drug-Eluting Bead—A Safety and Feasibility Pilot Study in a Rabbit Liver Cancer Model

The purpose of this article is to evaluate feasibility and safety of the cancer targeting (radio)-chemoembolization drug-eluting bead (TRCE-DEB) concept drug SW43-DOX-L-NETA(89Y) DEB for the intra-arterial treatment of VX2 rabbit liver tumors. The treatment compound comprises of the sigma-2 receptor ligand SW43 for cancer targeting, doxorubicin (DOX), and 89yttrium (89Y) as nonradioactive surrogate for therapeutic (yttrium-90, lutetium-177) and imaging (yttrium-86) radioisotopes via the chelator L-NETA. Ten New Zealand white rabbits with VX2 tumor allografts were used. SW43-DOX-89Y was synthesized, loaded onto DEB (100 μL; 100-300 μm), and administered intra-arterially in six rabbits at increasing doses (0.2-1.0 mg/kg). As controls, two rabbits each received either doxorubicin IV (0.3 mg/kg) or no treatment. Consecutive serum analysis for safety and histopathological evaluation after sacrifice were performed. One-Way ANOVA incl. Bonferroni Post-Hoc test was performed to compare groups. Targeted compound synthesis, loading onto DEB, and intra-arterial administration were feasible and successful in all cases. Serum liver enzyme levels increased in a dose dependent manner within 24 h and normalized within 3 days for 0.2/0.6 mg/kg SW43-DOX-89Y loaded onto DEB. The two rabbits treated with 1 mg/kg SW43-DOX-89Y had to be euthanized after 3/24 h due to worsening general condition. Histopathological necrosis increased over time in a dose depended manner with 95-100% tumor necrosis 3-7 days post treatment (0.6 mg/kg). SW43-DOX-89Y loaded onto DEB can be formulated and safely administered at a concentration of 0.6 mg/kg. Loading with radioactive isotopes (e.g., 86yttrium/90yttrium/177lutetium) to synthesize the targeted radio-chemoembolization drug-eluting bead (TRCE-DEB) concept drug is feasible.

In vitro biologic efficacy of sunitinib drug-eluting beads on human colorectal and hepatocellular carcinoma—A pilot study

Purpose Sunitinib drug eluting beads (DEB) are a novel anti-angiogenic bead preparation for use in transarterial chemoembolization. However, systematic studies of sunitinib DEB’s effect on cancer cells have not been reported. Herein, we assess their direct biologic efficacy against carcinoma cell lines and correlate cell viability with drug release in vitro. Materials and methods Sunitinib-HCl (10mg/mL) in Milli-Q water was mixed with LC Bead® 300–500μm (Biocompatibles UK Ltd.). Loading and release were assessed by measurement of drug UV absorbance using UV-visible spectrophotometer. Viability of human colorectal cancer (CRC, HCT116 and HT29) and hepatocellular carcinoma (HCC, HepG2) cells upon exposure to sunitinib DEB was measured using a bioluminescent assay. Drug concentration during exposure was quantified using HPLC. Results When added to cultured HepG2 cells, sunitinib DEB rapidly inhibited viability with a significant decrease observed within 1 hour of incubation. Viability of HCT116 and HT29 cells decreased relatively slower, with significant reductions observed after 8 and 24 hours, respectively. After 24 hours there was nearly complete inhibition of all three cell lines. There was no difference in viability observed between cells treated with 5 μl, 10 μL, or 20 μL of sunitinib DEB. HPLC analysis of the cell culture supernatant demonstrated saturation of the cell medium within approximately 4 hours for each amount added, with sunitinib achieving a final concentration of 17.61 μM (SE ±1.01). Conclusions Sunitinib can be efficiently loaded to and released from LC beads, and the resulting sunitinib DEB demonstrate strong in vitro inhibition of human CRC and HCC cells.

Abstract 870: Radiation-induced PD-L1 upregulation can be detected by Zr-89-PD-L1 PET/CT in the tumor micro-environment of murine HPV positive HNSCC model and melanoma model

Background: Radiation therapy (RT) can induce upregulation of programmed death ligand 1 (PD-L1) on tumor cells or myeloid cells, which might affect the response to RT with or without anti-PD-1/PD-L1 blockade. RT-induced PD-L1 expression during therapy could be a predictive marker for the therapy response, however, serial biopsies to monitor its distribution may be unreliable because its expression is heterogeneous. Therefore, non-invasive imaging of tumor PD-L1 expression could be more helpful. Materials and Methods: Two different murine tumor models (MEER and B16F10) were established in two locations, at the back of the neck and at the right flank of C57BL/6 mouse. Then fractionated RT (2Gyx4 or 2GyX10) with or without anti PD-1 therapy was delivered only to the neck tumor. PD-L1 expression was measured by PET/CT and biodistrubtion with Zr-89-DFO-anti-mouse PD-L1 monoclonal Ab (clone 10F.9G2) and the results were corroborated by flow cytometric analysis and immunohistochemistry. PET/CT imaging and biodistributions were performed 48 or 96 hours after tracer-injection. The change of PD-L1 expression on irradiated neck tumor was evaluated using non-irradiated flank tumor as a control. Endothelial cell morphology in tumor vessels was also analyzed by CD31 staining to determine whether RT-induced increased permeability of tumor vessels might result in increased non-specific tracer accumulations in the irradiated tumors. Results: PET/CT imaging and biodistribution study of ex-vivo tracer uptake values demonstrated significant increased tracer uptake in irradiated neck tumor compared to non-irradiated flank tumor in either case of MEER and B16F10. Tracer uptakes in the spleens were significantly decreased if high dose of non-labeled anti-mouse PD-L1 monoclonal Ab was given in advance before tracer-injection, which indicates the tracer is fully functional. Flow cytometry and immunohistochemistry showed PD-L1 upregulation in both irradiated tumors corroborating PET/CT imaging of RT-induced PD-L1 upregulation. Flow cytometry also revealed RT-induced PD-L1 upregulation on myeloid cells is more prominent than that of tumor cells in both MEER and B16F10. CD31-positive staining in the irradiated tumors was not different from that of non-irradiated tumors, which shows RT did not induce alterations in tumor blood vessels. PD-L1 upregulation of MEER was not seen unless it was performed 2GyX10 RT, however, that of B16F10 could be seen at an earlier time-point of 2GyX4. Combination therapy of anti PD-1 with RT did not show further PD-L1 upregulation compared to RT without anti PD-1 therapy. Conclusion: RT-induced PD-L1 upregulation in different tumor models of MEER and B16F10 was identified by PET/CT using Zr-89 labeled PD-L1 monoclonal Ab and its validity was corroborated by flow cytometric analysis and immunohistochemistry. Citation Format: Masahiro Kikuchi, Raghvendra M Srivastava, David A Clump, Julio A Diaz-Perez, Lingyi Sun, Dexing Zeng, W. Barry Edwards, Carolyn J Anderson, Robert L Ferris. Radiation-induced PD-L1 upregulation can be detected by Zr-89-PD-L1 PET/CT in the tumor micro-environment of murine HPV positive HNSCC model and melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 870. doi:10.1158/1538-7445.AM2017-870