Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response against the AH1 tumor rejection antigen

The possibility to cure immunocompetent mice bearing murine CT26 colorectal tumors using cytokine-based therapeutics allows to study the tumor rejection process at a molecular level. Following treatment with L19-mIL12 or F8-mTNF, two antibody fusion proteins which preferentially concentrate a murine cytokine payload at the tumor site, CT26 tumors could be cured in a process that crucially relies on CD8+ T cells. In both settings, the AH1 peptide (derived from the gp70 envelop protein of murine leukemia virus) acted as the main tumor rejection antigen and ~50% of CD8+ T cells in the tumor mass are AH1-specific after therapy. In order to characterize the clonality of the T cell response after successful antibody-cytokine immunotherapy, we isolated CD8+ T cells from tumors and submitted them to T cell receptor (TCR) sequencing. As expected, different TCR sequences were found in different mice, as these molecules originate from a stochastic rearrangement process. CD8+ T cells featuring the ten most abundant TCR sequences represented more than 60% of total CD8+ T cell clones in the tumor mass, but less than 10% in the spleen. Looking at sorted CD8+ T cells from individual animals, AH1-specific TCRs were consistently found among the most abundant sequences. Collectively, these data suggest that the antitumor response driven by two different antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.