Abstract PD13-08: An ENT2-dependent, cell-penetrating, and DNA-damaging lupus autoantibody crosses the blood-brain barrier to target breast cancer brain metastases
Authors: James E Hansen, Shenqi Zhang, Anupama Shirali, Christopher May, Benedette Cuffari, Valentina Dubljevic, James A Campbell, Jiangbing Zhou
Journal: Cancer Research (2021)

Abstract Introduction: Antibody-based immunotherapy has potential to change paradigms in the management of breast cancer brain metastases, if the blood-brain barrier (BBB) can be overcome. The autoimmune disease systemic lupus erythematosus offers an unexpected new approach to this problem. 3E10 is a lupus anti-DNA autoantibody that localizes to DNA at tumors, penetrates cells via the ENT2 nucleoside transporter, inhibits DNA repair, and is synthetically lethal to BRCA1/2 or PTEN-deficient cancer cells with impaired homologous recombination (HR). Breast cancer brain metastases exhibit increased HR defects and PTEN loss compared to primary breast tumors, and ENT2 expressed in brain endothelial cells (BECs) regulates nucleoside flux at the BBB. Further, 3E10 has previously delivered cargo protein to ischemic brain. We hypothesized that ENT2 may facilitate transport of 3E10 across the BBB, and that 3E10 could be used to treat breast cancer brain metastases. Deoxymab-1 (also known as PAT-DX1 or DX1) is a re-engineered and optimized fragment of 3E10 in pre-clinical development for use against HR-deficient tumors. In the present study we examined the ability of DX1 to cross the BBB and suppress breast cancer brain metastases. BBB study results: ENT2-dependent transport of DX1 across the BBB was evaluated in vitro and in vivo. DX1 penetrated hCMEC/D3 BECs and crossed from apical to basolateral chambers in an hCMEC/D3 transwell model of the BBB. Integrity of the model was confirmed by measuring transendothelial electrical resistance and demonstrating the barrier prevented movement of control protein into the basolateral chamber. Expression of ENT2 in hCMEC/D3 cells was confirmed by immunostaining, and the ENT2 inhibitor dipyridamole (DP) inhibited both penetration by DX1 into the cells and its transport across the BBB transwell model. Immunodeficient mice with orthotopic GBM tumors were treated with IV and IP control buffer (n=2), IV DX1 (20 mg/kg) and IP control buffer (n=4), or IV DX1 (20 mg/kg) and IP DP (ENT2 inhibitor, 70 mg/kg) (n=4). DX1 was labeled with Alexa Fluor 750 (AF750) to allow detection by IVIS. Twenty-four hours after treatment, mice treated with DX1 in the absence of DP exhibited strong AF750 signal in the brain correlating to tumor. Co-treatment with DP reduced uptake of DX1 into the brain tumors by ~78% (P&amp;lt;0.001). These findings are consistent with DX1 crossing the BBB and localizing into brain tumors in an ENT2-dependent manner. Efficacy study results: The 231-BR brain-seeking subclone of the MDA-MB-231 triple negative breast cancer cell line exhibits PTEN loss relative to parental cells. DX1 penetrated and killed 231-BR cells in vitro. For in vivo testing, brain metastases were generated in immunodeficient mice by intracardiac injection of 231-BR cells engineered for expression of luciferase. Brain metastases were confirmed by IVIS one week later, and then mice were treated with IV control buffer (PBS, n=7) or DX1 (20 mg/kg, n=7). In separate studies DX1 was delivered as a single cycle or as four consecutive cycles, with one cycle defined as control or DX1 3X/week. DX1 significantly suppressed tumor growth, evidenced by weekly IVIS. At week 5 brain radiance efficiencies (x105) in the single cycle study in control and DX1-treated mice were 264.8±72.0 and 71.9±31.3 (P&amp;lt;0.04), and 320±66 and 20.2±8.5 (P≤0.01) in the four-cycle study. One cycle of DX1 yielded a non-significant increase in median survival from 30 to 35 days (P=0.42). Four cycles of DX1 had greater impact, with median survival increased by 14 days (from 31 to 45) (P&amp;lt;0.002). DX1 was not associated with nonspecific toxicity. Conclusion: Our findings support an ENT2-mediated mechanism of BBB penetration by DX1 and establish proof of concept for use of a DNA-targeting autoantibody against breast cancer brain metastases. Citation Format: James E Hansen, Shenqi Zhang, Anupama Shirali, Christopher May, Benedette Cuffari, Valentina Dubljevic, James A Campbell, Jiangbing Zhou. An ENT2-dependent, cell-penetrating, and DNA-damaging lupus autoantibody crosses the blood-brain barrier to target breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-08.