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Abstract
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.
References
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
CBD-TNFa binds to collagen while remaining bioactive (A) Schematic of WT-TNFa and fusion of CBD to murine TNFa with glycine-serine (GGGS) linker, (B) SDS-PAGE of WT-TNFa and CBD-TNFa under reducing (R) and non-reducing (IN) conditions, with expected single bands with a shift in weight of ~20KDa for the CBD-TNFa fusion protein, indicated addition of CBD domain. (C) ELISA binding curves of WT and fusion protein binding to murine TNF-receptor 1 (TNRF1), Kd values are average of three independent experiments run in duplicates, mean ±SEM. (D) ELSA binding curves of WT-TNFa (i) and CBD-TNFa (ii) against purified collagen III. Kd values are average of two independent experiments run in duplicates, mean ± SEM
CBD-TNFa drives complete remission and improves survival in EMT6 bearing mice (A) Tumour growth curves of Balb/c mice (female, 8–12wk) bearing 5×105 EMT6 breast cancer cells subcutaneously on the back skin, were treated with PBS (n-5) 2ug CBD-TNFa (n=5) or 5ug CBD-TNFa (n=4) via intravenous injection on day 5 and day 9. Average growth curves (A) and individual growth curves (B) are shown - where 3/5 and 3/4 animals demonstrated CR (complete response) with 2ug CBD-TNFa and 5ug CBD-TNFa, respectively. Data from single experiment, presented as average ± SEM. (C) Survival curve of Balb/c mice (female, 8–12wk) bearing 5x105 EMT6 breast cancer cells subcutaneously on the back skin, treated with PBS (n=5), 2ug WT-TNFa (n=5)*, 5ug WT-TNFa (n=5)*, 2ug CBD-TNFa (n=5, TNFa molar equivalent) or 5ug CBD-TNFa (n=4, TNFa molar equivalent) via intravenous injection on day 5 and day 9. *, WT treated groups were culled after administration of second dose due to toxicity humane endpoint criteria being met, Statistical analysis was performed using a log-rank (Mantel-Cox) test using GraphPad prism (v9.5.1).
CBD fusion proteins shows favourable toxicity profile compared to WT-TNFa (A) Body weight change of EMT6 bearing mice treated with PBS (n=5), 2ug WT-TNFa (n=5), 5ug WT-TNFa (n=5), 2ug CBD-TNFa (n=5, TNFa molar equivalent) or 5ug CBD-TNFa (n=4, TNFa molar equivalent) via IV injection normalised to first treatment (day 5). Average ± SEM (B) plasma IL6 levels from blood taken 2 hours post first treatment with either PBS (n=6) 2ug WT-TNFa (n=6), 5ug WT-TNFa (n=6), 2ug CBD-TNFa (n=5, TNFa molar equivalent) or 5ug CBD-TNFa (n=6, TNFa molar equivalent), quantified using murine IL6 ELISA kit. Average ± SEM, one-wav ANOVA was performed using Graphpad prism (p<0.05).
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