Balancing the Dual Mechanisms of Anti-VISTA mAbs for Antitumor 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 (mAb), 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 antitumor efficacy in the CT26 syngeneic model, whereas Mu61 is ineffective despite having a slightly higher affinity for mVISTA than Vs22. Although 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 nonidentical 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 antitumor efficacy, in contrast to the inactive Mu61. Mechanistic studies revealed that Vs22's antitumor 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 antitumor 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 antitumor immunity and supports the clinical translation of VISTA-targeting therapeutics.