Background Tumour Necrosis Factor (TNF) is a pro-inflammatory cytokine capable of activating anti-cancer immune cell responses and directly inducing cancer cell necrosis.1 Although a very potent molecule its application as anti-cancer therapeutic has been hampered by severe toxicity, which is often characterised by cytokine release syndrome (CRS). To overcome the toxicity challenge and harness its therapeutic value, TNF must be localised to the tumour to minimise toxicity. One strategy is to use tumour extracellular matrix proteins such as collagen, as a reservoir, and exploit the leaky nature of tumour vasculature within the tumour microenvironment to specifically deliver therapeutic payloads. Our lab has previously shown that recombinant fusion of a collagen-binding domain (CBD), based on the A3 domain from blood protein von Willebrand factor (VWF), facilitates the localised delivery of cytokine payloads to the tumour microenvironment.2 Encouraged by this, we have fused the CBD domain to TNFa, and hypothesise that this fusion protein would show a favourable toxicity profile compared to WT-TNFa.
Methods Recombinant mouse TNFa and a CBD fusion version were produced using a transient mammalian protein production platform, purified using a histidine affinity column and size exclusion chromatography, then characterised by SDS-PAGE (figure 1A,B). Proteins were functionally characterised in vitro using binding ELISAs against collagen III and TNF-receptor 1 (TNFR1) (figure 1C,D). The efficacy and toxicity profiles of the fusion proteins were evaluated in vivo using a murine breast cancer model (figures 2 and 3).
Results Here we have shown that CBD-TNFa fusion proteins can be successfully produced, and retain their ability to bind to TNFR1 and can bind to collagen III - figure 1. Furthermore, in a murine breast cancer model intravenous administration of CBD-TNFa drives complete remission (3/5 and 3/4 mice, with 2 µg and 5ug CBD-TNFa respectively) (figure 2). Crucially, we observe a remarkable reduction in toxicity compared to WT-TNFa as indicated by minimal weight loss and reduction in plasma interleukin-6 (a cytokine which is strongly upregulated during CRS) (figure 3).
Conclusions The fusion protein remains biologically functional and, initial results, demonstrate a remarkable improvement in the toxicity profile of TNFa in EMT6 bearing mice. CBD-TNFa may open a new avenue for clinical translation of TNFa as an intravenously injected therapeutic.
Waters JP, Pober JS, Bradley JR. Tumour necrosis factor and cancer. In Journal of Pathology 2013;230(3):241–248. https://doi.org/10.1002/path.4188
Mansurov A, Ishihara J, Hosseinchi P, Potin L, Marchell TM, Ishihara A, Williford JM, Alpar AT, Raczy MM, Gray LT, Swartz MA, Hubbell JA. Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours. Nature Biomedical Engineering 2020;4(5):531–543. https://doi.org/10.1038/s41551–020-0549–2
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