Optimization of TNFR2-selective agonists with immunomodulatory properties : safety studies to support clinical development

Abstract

Tumor Necrosis Factor (TNF) is a cytokine playing a major role in immune regulation. It exists as a trimeric protein, either soluble (sTNF) or transmembrane form (tmTNF). TNFa can bind selectively to two transmembrane receptors: Tumor Necrosis Factor Receptor 1 (TNFR1) or 2 (TNFR2). While sTNF can only bind to TNFR1, tmTNF can bind to both receptors. TNFR1 is commonly found on most cell types, whereas TNFR2 is mostly expressed on the surface of epithelial, endothelial, and immune cells. Those receptors induce opposite effects upon activation. TNFR1 activation leads to inflammatory response, apoptosis, and neurodegeneration, while TNFR2 promotes tissue homeostasis and neuroregeneration. Because of those antagonistic effects, TNF and TNFRs are considered as promising targets. Therapeutic approaches using TNF inhibitors have been developed in the context of auto-immune and inflammatory diseases. Unfortunately, this global TNF blockade often leads to detrimental side effects, with limited responsiveness and an increase of infections. The genetic engineering of TNFR2-selective agonist fusion proteins represents a promising target in immunomodulatory therapy development. In this project, TNFR2 selective agonist molecules, mimicking tmTNF were developed. It consists of a TNFR2-specific mutant single chain TNF trimer merged to an effector-deficient Fc domain. This study focuses on optimizing the human TNFR2-selective agonist and evaluating biosafety for further clinical development. Functional bioactivity assessments of the TNFR2-selective agonists were conducted to evaluate receptor selectivity, bioactivity, and potential cross-reactivity among different mammalian species. NfkB reporter cell lines were developed to further the bioactivity of the molecules. In vivo studies examined TNFR2 expression in both wild-type and humanized TNFR2 C57BL/6J mice, along with pharmacokinetic profiles and tolerance of murine and human TNFR2 selective agonists. This project contributes to understand TNFR2 selective agonist fusion proteins, providing valuable perspectives for their clinical development in immunomodulatory therapies.