ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody
Zahra Rattray, Gang Deng, Shenqi Zhang, Anupama Shirali, Christopher K. May, Xiaoyong Chen, Benedette J. Cuffari, Jun Liu, Pan Zou, Nicholas J.W. Rattray, Caroline H. Johnson, Valentina Dubljevic, James A. Campbell, Anita Huttner, Joachim M. Baehring, Jiangbing Zhou, James E. Hansen
Zahra Rattray, Gang Deng, Shenqi Zhang, Anupama Shirali, Christopher K. May, Xiaoyong Chen, Benedette J. Cuffari, Jun Liu, Pan Zou, Nicholas J.W. Rattray, Caroline H. Johnson, Valentina Dubljevic, James A. Campbell, Anita Huttner, Joachim M. Baehring, Jiangbing Zhou, James E. Hansen
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Research Article Oncology

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody

Abstract

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Authors

Zahra Rattray, Gang Deng, Shenqi Zhang, Anupama Shirali, Christopher K. May, Xiaoyong Chen, Benedette J. Cuffari, Jun Liu, Pan Zou, Nicholas J.W. Rattray, Caroline H. Johnson, Valentina Dubljevic, James A. Campbell, Anita Huttner, Joachim M. Baehring, Jiangbing Zhou, James E. Hansen

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Figure 1

DX1 penetration into human brain endothelial cells is dependent on ENT2.

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DX1 penetration into human brain endothelial cells is dependent on ENT2....
(A) Illustrated evolution of 3E10 into DX1. 3E10 was isolated from the MRL/lpr lupus mouse model. A 3E10 single chain variable fragment (scFv) with D31N mutation in the heavy chain variable domain complementarity determining region 1 (VH CDR1) was previously shown to have higher affinity for DNA and efficiency of cellular penetration compared with the original 3E10. 3E10 D31N di-scFv has greater impact on the DNA damage response and synthetic lethality to PTEN-deficient cancer cells due to its increased avidity for DNA compared with the scFv. The 3E10 D31N di-scFv was humanized, deimmunized, and CDR-optimized to yield DX1, which is now in development for testing in clinical trials (8, 17). (B) DX1 penetrates into brain endothelial cells in an ENT2-dependent manner. hCMEC/D3 cells transfected with control or ENT2-targeting siRNA were treated with control buffer or DX1 alone, and then stained to detect DX1 penetration. Representative images are shown. Scale bar: 30 μm. (C) Control IgG shows minimal uptake into brain endothelial cells. hCMEC/D3 cells were treated with 0–10 μM of a control IgG (specifically an anti-PD1 antibody) and stained to detect uptake of IgG. Representative images are shown. Scale bar: 30 μm. (D) The ENT2 inhibitor DP interferes with DX1 penetration into brain endothelial cells. hCMEC/D3 cells were treated with control buffer, DX1, or DX1 + 50 μM DP and stained for DX1. Representative images are shown. Scale bar: 30 μm. These data demonstrate ENT2-dependent penetration by DX1 into hCMEC/D3 cells.