Abstract: FR-PO0789
Exploring SLIT2 as a Target Engagement Biomarker for a Novel Podocytopathy Therapy
Session Information
- Glomerular Diseases: Cell Homeostasis and Novel Injury Mechanisms
November 07, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Patel, Yash P, Boston Medical Center, Boston, Massachusetts, United States
- Dabas, Aarushi, Boston Medical Center, Boston, Massachusetts, United States
- Fan, Xueping, Boston Medical Center, Boston, Massachusetts, United States
- Liaw, Easton J., Boston Medical Center, Boston, Massachusetts, United States
- Kumar, Sudhir, Boston Medical Center, Boston, Massachusetts, United States
- Salant, David J., Boston Medical Center, Boston, Massachusetts, United States
- Waikar, Sushrut S., Boston Medical Center, Boston, Massachusetts, United States
- Lu, Weining, Boston Medical Center, Boston, Massachusetts, United States
Background
ROBO2/SLIT2 signaling destabilizes podocyte focal adhesions and attachment to the GBM. The SLIT2 inhibitor ROBO2-Fc enhances podocyte adhesion and protects animal models from podocyte injury in a dose-dependent manner. A phase II clinical trial of ROBO2-Fc in FSGS revealed the need for a sensitive assay of target engagement to establish the optimal therapeutic dose. To address this need, we have developed an ultrasensitive electrochemiluminescence immunoassay (ECLIA) for ROBO2-Fc target engagement with endogenous SLIT2 and have initiated preclinical studies to test it in animal models treated with ROBO2-Fc.
Methods
SLIT2 levels were quantified using ECLIA on the MESO Scale Discovery (MSD) platform. A SLIT2-specific capturing antibody was selected and confirmed with the SVZa neuron cell migration bioassay. Conditioned media from HEK293 cells transfected with SLIT2 recombinant cDNA were analyzed to verify the specificity of the SLIT2 ECLIA assay. The in vivo SLIT2 target engagement activity was investigated by measuring serum endogenous SLIT2 levels in mice treated with ROBO2-Fc at high and low doses.
Results
The SVZa neuron cell migration assay demonstrated strong functional binding affinity of the anti-SLIT2 antibody used in the ECLIA. Consistently across multiple SLIT2 ECLIA experiments, the standard curve produced a low limit of detection (LLOD) of SLIT2 ranging from 3 to 27 pg/mL. Analyses of conditioned media from HEK293 cells with and without transfected recombinant SLIT2 confirmed a high specificity of 95 ± 5% for the SLIT2 ECLIA. Mice treated with low (5 mg/kg) and high (25 mg/kg) doses of ROBO2-Fc exhibited a 40% and 80% decrease in serum SLIT2 levels, and high doses of ROBO2-Fc (25 mg/kg) also alleviated podocyte injury phenotypes in ILK podocyte-specific knockout mice, an animal model of podocytopathies.
Conclusion
We have developed an ultrasensitive target engagement SLIT2 ECLIA that reliably detects endogenous SLIT2 with high specificity and repeatability. High doses of ROBO2-Fc (25 mg/kg) inhibited endogenous SLIT2 by 80% and alleviated podocyte injury phenotypes in animal models of podocytopathies. We recommend that this assay be used to assess SLIT2 engagement by ROBO2-Fc in future clinical trials.
Funding
- NIDDK Support