Introductory statement: To overcome enzyme-mediated resistance mechanisms associated with the DNA alkylating agent, temozolomide (TMZ), we seek to develop a peptide-drug conjugate (PDC) for selective drug delivery in cells that express the somatostatin receptor subtype-2 (SSTR2). Material and methods: The clinically used somatostatin analog, 68Ga-DOTA-TOC, was used as a model for development of the SSTR2-targeted PDC. Synthesis was performed by (i) replacing DOTA with a multimodality chelator (MMC), (ii) attaching a modified TMZ analog to MMC, and (iii) conjugating the payload moiety to TOC on solid-phase. The resulting product, MMC(TMZ)-TOC, was radiolabeled with 67Ga using cation exchange chromatography methods optimized for 68Ga. Retention of SSTR2 binding was examined in cell lines with different expression level of SSTR2 with and without blocking doses of octreotide. A membrane acid wash was performed to evaluate internalization efficiency. The cytotoxicity of MMC(TMZ)-TOC was tested in IMR-32 cells that endogenously express SSTR2, and potency was compared to free-TMZ. An alkaline comet assay was then performed to assess the DNA-damaging properties of the PDC in the presence and absence of SSTR2 blocking. Unpublished results: MMC(TMZ)-TOC was efficiently produced with chemical purity >90% as shown by high-performance liquid chromatography (HPLC). Radiochemical purity following 67Ga labeling was >95% and indicates the feasibility of using the MMC to directly label the drug conjugates. Cell-based experiments showed that specific binding of the 67Ga-labeled PDC was similar to 67Ga-DOTA-TOC and correlated with SSTR2 expression. In HCT116-SSTR2 cells that overexpress SSTR2, 14.8±4.8% of 67Ga-MMC(TMZ)-TOC and 17.0±4.2% of 67Ga-DOTA-TOC were taken up by cells. Blocking the excess octreotide reduced binding to near background levels, illustrating receptor-mediated uptake of the PDC. Acid-washing demonstrated internalization of 67Ga-MMC(TMZ)-TOC after receptor-binding, indicating retention of the agonist properties of TOC after payload conjugation. Results from the cell cytotoxicity study demonstrated that the PDC inhibited cell growth in a dose-dependent manner that was similar to free-TMZ, with the IC50 values of 81.6 and 75.6 µM for free-TMZ and MMC(TMZ)-TOC, respectively. The comet assay revealed that MMC(TMZ)-TOC was effective in causing DNA damage in cells that express SSTR2, as shown by reduced cytotoxic effects when co-incubated with octreotide. Conclusions: We showed that a clinically used SSTR2-targeted radiopharmaceutical can be converted into a PDC that retains receptor-binding and internalization properties. We also showed that the cytotoxic effects of TMZ are maintained by the PDC. Based on these results, in vivo evaluation of MMC(TMZ)-TOC is warranted to assess its potential as a targeted therapy for NETs.

Citation Format: Solmaz AghaAmiri, Sukhen C. Ghosh, Lea Stitzlein, Servando Hernandez Vargas, Bruno Perlatti, Daniel M. Halperin, Ali Azhdarinia. A novel peptide-drug conjugate induces DNA damage in SSTR2-expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB120.