LAUSR

Abstract Background The E3 ubiquitin ligase Cbl-b is a key master immune checkpoint regulator that limits immune activation critical for anti-tumor immunity. We first reported that Cbl-b deficiency in mice confers spontaneous in vivo rejection of tumor cells. This checkpoint role in anti-tumor immunity was confirmed in multiple mouse and human studies. Further discovery efforts led to the advancement of APN401, an ex vivo human Cbl-b siRNA-based autologous cellular therapy, currently in clinical development in patients with advanced solid tumors. APN401 has been target validated and its preclinical efficacy was established in various mouse syngeneic tumor models. We present here the potent anti-tumor efficacy of APN401 immunotherapy of Cbl-b silenced murine T cells in a syngeneic MC38 colorectal tumor model. Methods T cells isolated from MC38 tumor-bearing donor C57Bl/6 mice were silenced ex vivo with APN401 murine Cbl-b specific siRNA vs control siRNA, adoptively transferred into MC38 tumor-bearing recipient mice, and tumor growth was monitored using an in vivo imaging system. Results Murine APN401 treatment resulted in a significant MC38 tumor growth inhibition of 63% (p = 0.008) vs controls after just a single dose application. Profound anti-tumor efficacy induced by Cbl-b-silenced T cells strongly correlated with enhanced production of TH1 cytokines IL-2 and IFN-γ. Furthermore, in vivo tracking of fluorescently labeled and silenced T cells revealed migration to relevant lymphoid organs and local tumor sites. Murine APN401 treatment was safe and well-tolerated similar to recent human APN401 clinical phase Ia study results in patients with advanced solid tumors. Conclusions In a model of murine colon cancer, Cbl-b-silencing induced vigorous in vivo anti-tumorigenic immune responses. The treatment modality was safe and well tolerated. These data provide direct preclinical proof of concept that siRNA-based silencing of Cbl-b provides a novel, effective and tunable approach as cellular immunotherapy. Targeting Cbl-b through human APN401 cell therapy is a promising novel therapeutic approach for the treatment of solid cancers, also highlighted by our fast-tracked global APN401 clinical development. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.