Abstract 4: Antitumor activity of DLX1008, a single chain antibody fragment binding to VEGF-A, in in vivo preclinical models of Kaposi sarcoma and glioblastoma
Authors: Eason Anthony B. , Sin Sang-Hoon , Szabó Emese , Phillips Douglas J. , Droste Miriam , Shamshiev Abdijapar , Dittmer Dirk P. , Weller Michael 
Published: August 2018

Abstract
Background:
Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of several cancers. Efficacy of VEGF inhibitors could be increased by improving affinity or tumor penetration, for example by reducing the size of the drug. Recently, the exceptionally stable anti-VEGF single chain variable fragment (scFv) brolucizumab (RTH258, ESBA1008, Novartis) completed phase 3 clinical trials in ophthalmology and was shown to be more efficacious than the larger aflibercept. The same molecule, named DLX1008, is currently in pre-clinical development in gliobastoma and Kaposi sarcoma (KS) by Cell Medica. KS is the most angiogenic cancer known to date. Carcinogenesis is induced by KS-associated herpesvirus (KSHV) and compromised of endothelial cells expressing high levels of VEGFR. KS is unique because angiogenesis (growth of non-tumor endothelial cells) as well as proliferation of the primary tumor cells are dependent on VEGF. No VEGF antagonist has yet been demonstrated to unequivocally improve overall survival in either KS or glioblastoma, which may be due to limited tumor penetration.
Methods:
Binding affinity to VEGF-A was determined by kinetic exclusion assay (KinExA).
For in vivo studies, we used the L1T2 model of KS, whereby KSHV-positive human cells (L1T2/ ATCC® VR-1802™) are implanted subcutaneously onto NGS mice, and the U87 model of glioblastoma, where human glioblastoma cells (U87/ ATCC® HTB-14™) are implanted subcutaneously or intracranially onto SWISS nude mice. Tumor growth was followed by caliper measurements and pathology evaluated by histochemistry or imaging.
Results:
DLX1008 binds to VEGF-A165 with a KD of 1.05 pM. Bevacizumab binds with a KD of 32.4 pM. Hence, the affinity of DLX1008 to human VEGF is around 30-fold tighter than that of bevacizumab.
DLX1008 reduced growth of L1T2 cells in vivo (p ≤ 0.007 by mixed effects model, n = 10) compared to vehicle control at 15 mg/kg once daily 5x/week and (p≤0.001, n = 10) in a second biological repeat. Imaging showed that the tumor died from the inside out in response to DLX1008. DLX1008 reduced the growth of U87 cells subcutaneously implanted in vivo (p ≤ 0.0021 by ANOVA, n = 10) and improved median survival of mice intracranially implanted with U87 cells (p ≤ 0.00026) compared to control scFv at 50 mg/kg twice daily. Staining of intracranial U87 tumors showed decreased CD31-positive blood vessel density as well as decreased p-VEGFR1 immunoreactivity in vascular structures and glioma cells.
Conclusions:
The high affinity, highly stable scFv DLX1008 shows highly reproducible anti-tumor activity in in vivo preclinical models of KS and glioblastoma. Efficacy was attributable to reduced angiogenesis. Its small size offers advantages in tissue penetration and distribution as compared to bevacizumab. DLX1008 is an excellent antibody to advance into clinical trials.
Citation Format: Anthony B. Eason, Sang-Hoon Sin, Emese Szabó, Douglas J. Phillips, Miriam Droste, Abdijapar Shamshiev, Dirk P. Dittmer, Michael Weller. Antitumor activity of DLX1008, a single chain antibody fragment binding to VEGF-A, in in vivo preclinical models of Kaposi sarcoma and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4.