Description

Eradication of experimental sarcomas by targeted delivery of TNF in combination with doxorubicin provides immunity against heterologous tumors

Philipp Probst^a, Janine Kopp^a, Annette Oxenius^b, Danilo Ritz^c, Tim Fugmann^c, Dario Neri*^a

^aInstitute of Pharmaceutical Sciences, ETH Zurich, Switzerland, ^bInstitute of Microbiology, ETH Zurich, Switzerland, ^cPhilochem AG, Otelfingen, Switzerland

Soft tissue sarcomas are a heterogeneous group of malignancies, which typically do not respond to doxorubicin, the first-line treatment of patients with metastatic disease.

We investigated the therapeutic activity of doxorubicin in combination with the immunocytokine F8-TNF in WEHI-164 sarcoma-bearing mice. The F8 antibody (specific to the extra-domain A of fibronectin) enables the selective delivery of the pro-inflammatory cytokine to the tumor neo-vasculature, causing hemorrhagic necrosis and a CD8+ T cell and NK cell dependent eradication of the tumor, as evidenced by in vivo depletion studies. Animals, which had been cured from the primary tumor, rejected subsequent challenges with the same cells, but also with heterologous C51 or CT26 colorectal tumor cells in a process that mainly depended on the action of CD8+ T cells. To further understand the CD8+ T cell-driven processes, we performed an exome sequencing to identify potential neo-epitopes and we used an immunocapture-LC/MS assay to characterize the MHC class I-bound peptides (“MHC class I peptidome”, with more than 4000 highly-confident peptide identifications). No neo-epitope could be identified in the MHC class I peptidome of the tumor cell line. However, all three tumors presented the AH1 peptide derived from the endogenous retroviral protein gp70. Tetramer-based analysis of spleen sections showed the presence of H-2L^d/AH1-reactive T cells in treated animals, suggesting their contribution to the rejection process. Sequence analysis of T cell receptors of CD8+ T cells revealed the presence of H-2L^d/AH1-specific T cells and an expansion of sequence diversity in treated mice. A human analogue of the fusion protein used in this study is about to start Phase III clinical trials in patients with metastatic soft-tissue sarcoma.