<div>AbstractPurpose:<p>Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity.</p>Experimental Design:<p><i>In vitro</i>, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. <i>In vivo</i> tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed.</p>Results:<p>The combination approach significantly increased viral replication and oncolysis <i>in vitro</i>, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed <i>in vivo</i> showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) T_Reg suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment.</p>Conclusions:<p>The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.</p></div>

<p>Supplemental Figure 1: Combination of XVir-N-31 and CDK4/6 inhibition increases oncolysis, viral particle formation associated by RB downregulation.</p>

<p>Supplemental Figure 4: Combination of XVir-N-31 and CDK4/6 inhibitor LEE011 induces an abscopal effect, induces monocyte infiltration and increases M1/M2 macrophage ratio in injected tumors.</p>

<p>Supplemental Figure 3: Characterization of tumor-infiltrating T cells at the end of experiment (A673 xenografts).</p>

<p>Supplemental Figure 4: Combination of XVir-N-31 and CDK4/6 inhibitor LEE011 induces an abscopal effect, induces monocyte infiltration and increases M1/M2 macrophage ratio in injected tumors.</p>