Abstract
Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 μg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.
Highlights
Cytokines are small proteins which modulate the activity of the immune system in health and in disease
We describe a depotentiated version of Tumor necrosis factor (TNF), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity
Our study was initially based on the identification of potential residues for the generation of TNF mutants which exhibit a decreased trimerization potential and, a reduced potency in solution
Summary
Cytokines are small proteins which modulate the activity of the immune system in health and in disease. Targeting an active cytokine to a tumor-associated antigen by means of a fusion with a suitable monoclonal antibody may represent a strategy for the enhancement of the cytokines’ therapeutic index [11,12]. Tumor necrosis factor (TNF) is a homotrimeric cytokine which shows direct cytotoxic activity on certain types of cancer cells [20], stimulates anti-tumor immunity [21,22], and may be active on the tumor endothelium, leading to intravascular blood coagulation [23] or to haemorrhagic necrosis [24]. We present the generation of a depotentiated version of Fibromun product, capable of inducing potent in vivo anti-tumor activity with reduced systemic toxicity. The use of engineered mutant versions of immunocytokines, like L19-TNFI97A, could represent a new avenue to develop products with ‘activity-on-demand’ properties
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