48 | wetenschapsdag 2023 Sessie 1d: Een Goede Fundering Voor de Chirurgische Lering x2 Auteurs D.Y. Gout, C.A.N. Sewnath, C.W. Tuk, A.E.H. Bentlage, N. Heemskerk, G. Vidarsson, M. van Egmond Abstract title Fusion of TNF-α to TrisomAb potentiates neutrophilmediated anti-tumor effects Background Neutrophils are an underestimated leukocyte subset in cancer immunotherapy. It is the most abundant leukocyte in circulation and possesses strong tumorkilling capabilities when stimulated with antibodies of the IgA isotype. Stimulation of IgA receptor FcαRI using a bi-specific antibody induces strong neutrophil recruitment to the edge of the tumor, but tumorinfiltration by recruited neutrophils is negligible. This strongly suggests an innate immune excluded tumor phenotype, comparable to the more well-known T-cell excluded phenotype; ‘cold’ tumors. Methods In order to break this innate immune exclusion, we have developed an antibody-cytokine fusion molecule consisting of the bi-specific FcαRI stimulator mentioned earlier and a TNF-α trimer. TNF-α has been shown to be able to affect the tumor microenvironment in various ways and is able to affect most (immune) cells. We expect the pleiotropic effects of TNF-α to synergize with the effects of the bi-specific antibody to increase the total efficacy. Results After successful production of the molecule was confirmed, binding assays showed that fusion of a cytokine did not impair binding affinities of the original antibody. Additionally, in vitro tumor killing using macrophages was shown to be unimpeded in ADCP assays, while tumor killing by neutrophils was increased. Using intravital imaging, we showed that treatment with the fusion molecule increases tumor infiltration and the occurrence of swarming behavior of neutrophils. Following these promising results, we are now investigating the in vitro effects of TrisomAb-TNF on the adaptive immune system as well as its effect on tumor growth and survival in vivo.
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