Abstract 6272: Redirecting tumor extracellular proteases to target radiosensitizer delivery

Abstract

Abstract Patients with locally advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and side effects severe. Drugs that sensitize tumors to radiotherapy have been tried to improve cure, but tumor specificity remains a challenge. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting utilizing peptide-based drug conjugates that split the responsibilities for targeting and radiosensitization into distinct molecular tasks. The tumor milieu possesses localizing beacons that can guide drug delivery using tumor cell surface receptors and microenvironment extracellular matrix metalloproteinases (MMPs). Here we applied peptide-based linkers to test for tumor selective drug delivery and radiosensitization by chemically conjugating the DNA damage checkpoint kinase 1/2 inhibitor AZD7762 to antibody to or cell penetrating peptides. AZD7762 governs DNA damage response and irradiated cell fate. We synthesized drug conjugates of AZD7762 and compared their anti-tumor efficacy in combination with ionizing radiation (IR). An antibody drug conjugate (ADC) was built by attaching AZD7762 to the anti-EGFR antibody cetuximab. Polycationic cell penetrating peptide drug conjugates were constructed by linking AZD7762 to a 9 amino acid repeat of D-arginine and keeping it bare (r9-AZD7762) or cloaking r9 within a MMP-2/9 triggered activatable cell penetrating peptide scaffold (ACPP-AZD7762). In contrast to receptor directed ADCs, ACPPs redirect extracellular tumor microenvironment proteases to guide drug delivery. Intact, ACPPs are charge neutral which blocks the conjugated drug from cell entry. Upon cleavage by tumor overexpressed MMPs, the ACPP scaffold releases drug conjugated polycationic peptide which is then internalized by cells. We found that while cetuximab-AZD7762 ADC had high affinity for EGFR overexpressing tumor cells and prolonged blood circulation in murine models, cetuximab-AZD7762 lacked anti-tumor efficacy. On the other extreme, naked r9-AZD7762 drug conjugate was indiscriminately cytotoxic and radiosensitized a panel of tumor cell lines in cell culture: CAL27, A549, PANC1 and HCT116. However, r9 cell penetrating peptides lacked tumor specificity when intravenously injected into mice. In contrast, MMP-2/9 sensitive ACPPs balanced tumor selectivity with drug efficacy. Intact, ACPPs held conjugated AZD7762 in a pro-drug inactive state that upon tumor microenvironment ACPP cleavage resulted in tumor selective drug accumulation and anti-tumor efficacy in combination with IR. Importantly, a control non-cleavable ACPP-AZD7762 did not improve tumor control with IR, demonstrating the necessity of ACPP cleavage to release active drug. These results highlight the utility of the ACPP technology platform to target amine containing radiosensitizing drugs to tumor tissues based on extracellular protease activity that overcome the limitations of ADC conjugated radiosensitizers. Citation Format: Dina V. Hingorani, Maria F. Camargo, Maryam A. Quraishi, Joseph Aguilera, Stephen R. Adams, Sunil J. Advani. Redirecting tumor extracellular proteases to target radiosensitizer delivery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6272.