Abstract 581: Peptide-based theranostics targeting ANTXR1 in cancer stroma
Authors: Li Shengjun , Choi Jin Yeong , Ha Hye Sook , Oh Yoo Joung , Lee Song-Gil , Choi Hongyoon , Im Hyung-Jun 
Published: April 2023

Background: ANTXR1 is one of the pancreatic ductal adenocarcinoma (PDAC) stroma-specific cell surface markers (Ref 1), which is also overexpressed in the stroma of various cancer types. Radiotheranostics refers to the combination of molecular imaging with targeted radionuclide therapy. Recent success of radiotheranostic agents such as 177Lu-DOTATATE and 177Lu-PSMA617 heralds the vibrant expansion of radiotheranostics. Herein, we sought to develop ANTXR1 targeting peptide as a cancer stroma-targeting radiotheranostic agent.

Methods: We selected A549 and NCI-H460 as ANTXR1 positive cell lines and NCI-H1437 and NCI-H358 as negative cell lines based on a proteomics database for cancer cell lines. ANTXR1-targeting peptide (PC01) was discovered using Chemistry-based Ultra-high-throughput Screening (CUSTM) platform. PC01 was conjugated with either fluorescence dye (Cy5-PC01) or chelator (DOTA-PC01). DOTA-PC01 was labeled with 68Ga. Flow cytometry and confocal microscopy were utilized to assess specific binding of the peptide. Subcutaneous tumor-bearing mice were intravenously injected with Cy5-PC01 or 68Ga-DOTA-PC01. In vivo/ex vivo fluorescence imaging (IVIS) and positron emission tomography (PET) imaging were performed.

Results: In western blot analysis, H460 and A549 demonstrated high levels of expression of ANTXR1, while H1437 and H358 showed negligible levels of expression, consistent with the proteomics database. The mean fluorescence intensity of H460 was significantly higher than that of H1437 after incubation with Cy5-PC01 in flow cytometry. In confocal microscopy, the two ANTXR1 positive cell lines demonstrated higher fluorescence signals compared to the two ANTXR1 negative cell lines, which indicates the specific binding of PC01 to ANTXR1. As for in vivo experiments, timepoints of image acquisition for both IVIS and PET were 0h, 1h, 2h, and 4h after injection of the peptide. In in vivo IVIS images, tumor uptake was more prominent in H460 tumors than in H1437 tumors. Furthermore, tumor/kidney uptake ratio and tumor/liver uptake ratio of Cy5-PC01 were significantly higher in H460 tumors compared to those of H1437 tumors (P &lt; 0.05, P &lt; 0.05, respectively). Radiolabeling efficiency of 68Ga-DOTA-PC01 was measured to be over 95% by thin-layer chromatography. Serial PET images of 68Ga-DOTA-PC01 demonstrated an initial intense renal uptake and subsequent fast renal clearance, which is a typical biodistribution of peptide-based radiotheranostic agents. Also, the tumor uptake was more pronounced in the H460 tumors than in the H1437 tumors.

Conclusion: We developed an ANTXR1 targeting peptide conjugated with DOTA for radiotheranostics. We found that PC01 demonstrated favorable biodistribution and specific in vitro/in vivo binding to ANTXR1, which prompts us to further optimize and develop a peptide-based ANTXR1 targeting radiotheranostic agent.

Ref 1. Cancer Res (2022) 82 (12_Supplement): 5007

Citation Format: Shengjun Li, Jin Yeong Choi, Hye Sook Ha, Yoo Joung Oh, Song-Gil Lee, Hongyoon Choi, Hyung-Jun Im. Peptide-based theranostics targeting ANTXR1 in cancer stroma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 581.