An experimental model of anti-PD-1 resistance exhibits activation of TGFß and Notch pathways and is sensitive to local mRNA immunotherapy

Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or β2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFβ and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFβ and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.