Balancing the Dual Mechanisms of Anti-VISTA mAbs for Anti-Tumor Activity: CD8+ T Cell Responses and Fc-dependent Depletion of VISTA-Expressing Immune Cells

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a negative immune checkpoint and an emerging target for cancer immunotherapy, yet its broad expression on myeloid cells and T cells has complicated therapeutic antibody development. Here, we report two monoclonal antibodies (mAbs), Vs22 and Mu61, which were engineered from a common parental antibody and are cross-reactive with both human VISTA (hVISTA) and mouse VISTA (mVISTA). Vs22 demonstrates anti-tumor efficacy in the CT26 syngeneic model, whereas Mu61 is ineffective despite having a slightly higher affinity for mVISTA than Vs22. While both mAbs block VISTA-ligand interactions and deplete VISTA⁺ tumor-infiltrating immune cells via NK cytotoxicity, they differed in several aspects: they recognize similar but non-identical epitopes; only Vs22 enhances cytokine production by CD8⁺ T cells; and Vs22 exhibits a better pharmacokinetic (PK) profile and greater VISTA endocytosis. This multifaceted profile likely underlies Vs22's anti-tumor efficacy, in contrast to the inactive Mu61. Mechanistic studies revealed Vs22's anti-tumor activity requires coordinated fragment antigen-binding (Fab)-mediated VISTA blockade and fragment crystallizable (Fc) effector function and Vs22 treatment leads to durable, tumor-specific immune memory. Notably, combining Vs22 with TABBY106, an agonistic anti-4-1BB antibody, produced synergistic anti-tumor effects across CT26, MC38, and EMT6 models, substantially outperforming monotherapy. Collectively, we identify Vs22 as a cross-species anti-VISTA mAb that achieves effective tumor control by simultaneously blocking VISTA signaling and engaging strong Fc-mediated cytotoxicity. The synergy between Vs22 and 4-1BB-stimulation highlights a promising combination strategy to enhance anti-tumor immunity and supports the clinical translation of VISTA-targeting therapeutics.