Resulting from intracellular translation of nanoparticle delivered mRNA, mRNA therapeutics offers an unparalleled opportunity to direct the expression of proteins to specific subcellular compartments. Many cytokines, especially the immunomodulatory Tumor Necrosis Factor (TNF) superfamily members, exist as both membrane and soluble proteins. Trimeric members of the TNF superfamily are known to be susceptible to protease cleavage resulting in their release from the membrane. Membrane and soluble versions of the immunomodulators may have different activity. Several studies have shown that membrane expressed TNFSF members more efficiently cross-link TNFSF receptors resulting in a stronger activation or inhibition signal. Moreover, the activity of membrane expressed proteins is confined to the cells that express the protein and the cells that interact with the protein expressing cell. Thus, directing the expression of immunostimulatory proteins to the membrane could be a transformative way to enhance the activity while mitigating the toxicity by limiting systemic exposure. In this study we utilized Nutcracker Therapeutics Inc. unique mRNA platform to develop a membrane stabilized version of tumor necrosis factor super family member 14, also known as LIGHT, with superior co-stimulatory activity and stability. LIGHT is an immunomodulatory cytokine that primarily acts on lymphocytes and innate immune cells through the cis or trans binding of HVEM or lymphotoxin beta receptor (LtbR). LIGHT has been implicated in multiple inflammatory diseases and is currently being evaluated in the development of immunotherapies against cancer. LIGHT stimulatory activity depends on the displacement of the inhibitory BTLA that is normally bound to HVEM and LtbR. Studies have shown that membrane bound LIGHT, but not soluble LIGHT, can effectively displace BTLA. In this study, we hypothesized that the advantageous activities of mRNA derived membrane expressed LIGHT could be further enhanced with an engineered LIGHT molecule that is less susceptible to membrane shedding. We made multiple variants of Engineered LIGHT where different regions of the putative protease sensitive sites were replaced with either a flexible or rigid linker. In short, we found that i) the soluble version of LIGHT can inhibit immune stimulatory capability of membrane expressed LIGHT; ii) membrane bound LIGHT enhances co-stimulation of antigen-specific T cells; iii) engineered LIGHT enhances membrane expression and increases stability; and iv) a design using a novel rigid linker to replace the putative protease sensitive region of LIGHT showed best overall activity. Here, we demonstrate the capability of our integrated platform to design and deliver an enhanced, membrane stabilized LIGHT molecule. This engineered transmembrane molecule represents a new class of drug uniquely enabled by nanoparticle delivered mRNA.

Citation Format: Adrienne Sallets, Weiqun Liu, Meredith L. Leong, Ruben Prins, Diane M. Da Silva, Daniel J. Fernandez, Colin J. McKinlay, Edward E. Lemmens, Chris S. Rae, Sudha Adusumilli, Ray Low, Srinivasa Bandi, Guna Kannan, W. Martin Kast, Samuel Deutsch, Ole A. Haabeth. Enhanced membrane stability of mRNA nanoparticle derived TNFSF14 results in superior T-cell and NK cell stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6536.