An Attenuated Targeted-TNF Localizes to Tumors In Vivo and Regains Activity at the Site of Disease

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.

The present and future of immunocytokines for cancer treatment

“…Similar to including point mutations to reduce antibody-mediated toxicity, this technique has also been successfully applied to cytokines [ 145 , 146 ]. These mutated cytokines are referred to as Activation-by-Targeting Cytokines (AcTakines).…”

Targeting the Blood–Brain Tumor Barrier with Tumor Necrosis Factor-α

“…Early findings in glioblastoma patients showed that L19-TNF monotherapy could decrease blood perfusion within the tumor, which was associated with increased tumor necrosis and T-cell infiltration [ 67 , 69 ]. Anti-CD13 antibodies have also been used as TNF vehicles [ 70 , 71 ]. An interesting strategy developed to reduce the systemic toxicity of antibody-TNF conjugates is based on the use of conjugates bearing mutations in the TNF moiety that decrease its affinity for TNF receptors, thereby reducing off-target effects on normal cells [ 70 , 71 ].…”

“…Anti-CD13 antibodies have also been used as TNF vehicles [ 70 , 71 ]. An interesting strategy developed to reduce the systemic toxicity of antibody-TNF conjugates is based on the use of conjugates bearing mutations in the TNF moiety that decrease its affinity for TNF receptors, thereby reducing off-target effects on normal cells [ 70 , 71 ]. These de-potentiated TNF versions, thanks to their specific accumulation on the target molecules, such as alternatively spliced EDB domain of fibronectin or CD13, can regain their activity on targeted cells, e.g., by local avidity-driven receptor binding.…”

“…These de-potentiated TNF versions, thanks to their specific accumulation on the target molecules, such as alternatively spliced EDB domain of fibronectin or CD13, can regain their activity on targeted cells, e.g., by local avidity-driven receptor binding. For example, administration of high doses (375 µg/kg) of a depotentiated version of L19-TNF (I97A) to mice bearing subcutaneous WEHI-164 fibrosarcomas showed a more potent anti-tumor activity, without apparent toxicity, than the wild-type product [ 71 ]. Similarly, daily injection of a CD13-specific VHH single-domain antibody fused to a mutated (Y86F) single-chain murine TNF (50 µg/mouse) could selectively activate the tumor neovasculature in a murine melanoma model without detectable toxicity [ 70 , 71 ].…”

“…For example, administration of high doses (375 µg/kg) of a depotentiated version of L19-TNF (I97A) to mice bearing subcutaneous WEHI-164 fibrosarcomas showed a more potent anti-tumor activity, without apparent toxicity, than the wild-type product [ 71 ]. Similarly, daily injection of a CD13-specific VHH single-domain antibody fused to a mutated (Y86F) single-chain murine TNF (50 µg/mouse) could selectively activate the tumor neovasculature in a murine melanoma model without detectable toxicity [ 70 , 71 ]. Although these approaches might reduce the systemic toxicity of conjugates, the antibody ligands, the linkers, the mutations, and the markedly high doses necessary to induce anti-tumor effects might negatively impact the immunogenicity of this class of compounds, a point that should be carefully considered.…”