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Background Immuno-oncology (IO) bispecific antibodies such as volrustomig (monovalent, bispecific anti-PD-1/-CTLA-4) have demonstrated promising activity in Phase 1 clinical trials and are being further investigated in multiple Phase 3 trials across solid tumor indications. We have sought to dissect the biological differentiation of volrustomig in nonclinical assays to support clinical positioning, though demonstrating combinatorial anti-CTLA4 + anti-PD1 activity presents challenges due to model limitations. However, the development of humanized mice may provide a reliable tool to investigate IO therapeutic effects on different components of the immune system by enabling long-term engraftment of functional multi-lineage hematopoietic cells.Methods In this study, we developed a CD34 +-humanized mouse tumor model (CD34-SGM3 mice) implanted with human esophageal tumor engineered with high affinity peptide (OE21V2) for a better recruitment and activation of immune cell in tumor. Therefore, from this model, we compare the therapeutic efficacy and pharmacodynamic effect of anti-PD-1 monotherapy and volrustomig. We developed a 25-marker spectral flow cytometry antibody panel to assess major immune cell lineage populations, the amount of T-cell activation and expression levels of immune checkpoint markers. In parallel, bulk RNA sequencing of tumor samples was conducted to characterize the genetic changes associated with the immune response following antibody treatment (figure 1).Results Our in vivo analysis revealed that 1) antigen recognition favors the recruitment of immune cells in tumors and this infiltration is potentiated with volrustomig in comparison with anti-PD-1 monotherapy, 2) high dimensional flow cytometry revealed that volrustomig gives rise to distinct infiltrating T cell subsets but alters myeloid recruitment in tumors and 3) transcriptomic analyses showed a distinct program triggered by volrustomig with significant up-regulation of genes encoding for IFNγ-signaling pathway and a specific set of transcription factors.Conclusions Overall, using in vivo tumor with high-affinity peptide allows to study how IO treatments impact tumor microenvironement and our data demonstrates that volrustomig therapeutic approach potentiates anti-tumor immune activity.Abstract 447 Figure 1Immune response and transcriptomic profiling of peptide-engineered esophageal tumors in IO-treated CD34+ humanized mice