Abstract 6085: Engineered anti-CD40 agonist antibody as a novel cancer neoantigen vaccine carrier

Abstract Somatic mutations in cancer cells lead to the expression of neoantigens that can be targeted by personalized vaccines utilizing patient’s own host immune system to fight against cancer. However, current personalized cancer vaccines have shown poor clinical outcomes due to the lack of effective delivery methods. To address the issues, our study aims to develop a novel vaccine platform that targets dendritic cells (DCs) and delivers neoantigen peptides more effectively by an anti-CD40 agonistic antibody expressing monovalent streptavidins (αCD40-mSAs). The agonistic anti-CD40 antibody will target and activate DCs, and the mSA compartment will allow the easy and fast loading of two biotinylated payloads (e.g. peptides and RNAs) on each vaccine carrier. CHO cell-based recombinant protein production method was used to biosynthesize αCD40 and αCD40-mSAs. Cloning vectors were reconstructed using mouse IgG2a, incorporating peptide sequences from the anti-mouse CD40 agonist antibody (clone FGK45) and monovalent streptavidin. For the biophysical properties of produced αCD40-mSAs, Surface Plasmon Resonance (SPR) for mouse CD40 and fluorescence polarization for biotin binding of mSAs have been used. Biotin- ovalbumin (OVA) peptide loaded αCD40-mSAs were subcutaneously injected into the footpad of the mice, and the collected lymph nodes were imaged with confocal microscopy and checked subtype of immune cells using flow cytometry. Antigen-specific T-cell responses were evaluated seven days post-injection by collecting lymph nodes and spleen. We successfully produced αCD40-mSAs, confirmed by increased molecular weight on the light chain expressing monovalent streptavidin. Binding assays demonstrated high-affinity interactions with mouse CD40 and effective biotin binding by the mSA moiety. In vivo targeting studies using fluorescently labeled OVA peptides revealed that αCD40-mSAs significantly enhanced peptide uptake in lymph nodes, particularly by cDC1 cells and macrophages. There was a notable increase in antigen-specific T cells, with an increase of OVA-specific CD8+ T cells and increased production of cytokines IL-2 and IFN-γ upon restimulation when OVA peptides were loaded onto αCD40-mSAs. In conclusion, αCD40-mSAs represent an effective delivery platform for personalized cancer vaccines by targeting DCs and improving neoantigen peptide uptake, offering a promising strategy to boost vaccine efficacy in cancer immunotherapy. Citation Format: Dahee Jung, Xiaoying Cai, Wonkyung Oh, Seung-Oe Lim, Steve Lee. Engineered anti-CD40 agonist antibody as a novel cancer neoantigen vaccine carrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6085.