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Background ATOR-4066 is a bispecific antibody developed using Alligator’s RUBY™ platform, targeting CD40 on antigen-presenting cells (APCs) and CEACAM5, a tumor-associated antigen expressed on tumor cells and tumor-derived material (e.g., exosomes, debris). 1 2 By simultaneously engaging CD40 and CEACAM5, ATOR-4066 promotes localized activation of dendritic cells (DCs) and macrophages, enhances uptake of tumor antigens, and facilitates cross-priming of neoantigen-specific T cells. In vivo, ATOR-4066 demonstrates superior anti-tumor efficacy compared to CD40 monospecific antibodies. While initial tumor control is T cell-independent, long-term efficacy requires functional T cell responses. Here, we further elucidate the mechanism of action of ATOR-4066, focusing on immune activation and extracellular matrix (ECM) remodeling.Methods Human CD40 transgenic mice bearing CEACAM5-transfected MC38 tumors were treated intraperitoneally with ATOR-4066 or vehicle on days 6 and 10 post-inoculation. Tumors were harvested 24 hours after the second dose for analysis. Whole tumors and isolated immune compartments (CD45 +Ly6G–) were subjected to bulk RNA sequencing followed by normalization of raw gene counts analysis of differentially expressed genes. Subsequently, over-representation analysis was performed on differentially expressed genes and signature scores were generated for immune cell populations and extracellular pathways.Results In the tumor immune compartment, ATOR-4066 treatment downregulated genes associated with ECM organization (GO:0030198). Whole tumor analysis revealed upregulation of ECM disassembly pathways (GO:0022617). ECM disassembly signatures positively correlated with infiltration of immune cells, including neutrophils. ATOR-4066 also modulated matrix metalloproteinase (MMP) gene expression, notably upregulating anti-fibrotic MMPs such as Mmp12 and Mmp13 within intratumoral immune cells.Conclusions These findings demonstrate that ATOR-4066 remodels the tumor microenvironment by promoting ECM disassembly and immune activation. Together, this further supports development and clinical testing of ATOR-4066 in CEACAM5-expressing tumors.References Hägerbrand K, et al. Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies. J Immunother Cancer 2022;10(11).Nyesiga B, et al. RUBY(R) - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties. MAbs. 2024;16(1):2330113.Ethics Approval All animal experiments were performed according to approval from the Malmö/Lund Animal Ethics Committee, No: 16318/2019 and 18602/2023 in line with Arriva guidelines.