(T1130-02-10) Positively Charged Surface in LIGHT Trimers Fused with Fc, a Ligand for Lymphotoxin-b Receptor (LTbR) and HVEM, Caused a Non-specific Interaction with Cells Leading to Fast Systemic Clearance in Mice

Purpose: The lymphotoxin-b receptor (LTbR) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. The agonistic activation of LTbR has been shown to suppress tumor growth and enhance tumor chemosensitivity. More recently, expression within a tumor of a ligand for LTbR called LIGHT, which also interacts with HVEM (herpesvirus entry mediator) and DCR3 (decoy receptor 3), was shown to trigger an immunomediated tumor response. As such, a molecule utilizing two mouse LIGHT trimers fused with Fc (muLight-Fc) to extend systemic exposure was developed as a potential surrogate in mice for a human anti-tumor agent. However, this molecule showed fast clearance in mice and bears extended positively charged regions on its surface. In this study, we investigated the potential impact of the positively charge region on the fast clearance of muLIGHT-Fc in vitro and in vivo, and support the development of this novel format as a potential therapeutic agent.

Methods: We utilized two approaches to determine the impact of charged surface on cell interactions. The first one was to mutate two amino-acids from muLIGHT-Fc to reduce the positive charge (charge variant). The second one was to co-incubate of heparin with muLIGHT-Fc to interrupt charge interaction. Briefly, both muLIGHT-Fc and its charge variant was radiolabeled with [¹²⁵I], and then incubated in mouse blood and plasma to determine its stability and potential red-blood cells (RBC) partitioning. The radiolabeled molecules were also assessed for binding to human umbilical vein endothelial cells (HUVECs) which have low expression of LTbR in the presence or absence of heparin. Samples from plasma incubation were analyzed for any complex formation (with plasma protein) or degradation using size-exclusion high-performance liquid chromatography (SE-HPLC) while incubated blood samples were separated into cell pellet and plasma fractions to determine RBC partitioning using a gamma-counter. Similarly, following incubation of HUVECs with radiolabeled molecules, cells were washed and harvested for the quantification of radioactivity using gamma-counter. Data were normalized by cell numbers.

Results: Following incubation in plasma, neither muLIGHT-Fc nor its charge variant showed any sign of complex formation or degradation as determined by SE-HPLC. In contrast, incubation in blood revealed the muLIGHT-Fc was highly bound to blood-cells as compare to its charge variant and a control IgG₁ (40% in muLIGHT-Fc vs 1% of radioactivity in variant and control IgG). Consistently, HUVEC binding assay demonstrated that muLIGHT-Fc had more than 10-fold higher binding than its charge variant. Co-incubation with heparin significantly reduced the non-specific binding of muLIGHT-Fc to HUVECs. These results demonstrate that the positively charged surface can cause strong non-specific interaction with RBCs and vascular endothelial cells, which may lead to the fast clearance. An in vivo mouse study is currently underway to test the impact of heparin co-infusion on the PK of muLIGHT-Fc.

Conclusion: This study highlights the importance of molecule surface charge in driving PK. Characterization of these properties at early stage using various approaches is very valuable for the selection of molecules with appropriate PK for further development.