Background: Despite significant progress in cancer immunotherapy in recent years, resistance to existing immune checkpoint therapies (ICT) is common. V-domain Ig suppressor of T cell activation (VISTA), a predominantly myeloid immune checkpoint regulator, represents a promising therapeutic target due to its role in suppressing proinflammatory antitumor responses in myeloid-enriched tumor microenvironments. However, uncertainty around the cognate VISTA ligand has made the development of effective anti-VISTA antibodies challenging. The expression of VISTA on normal immune cell subtypes argues for a neutralizing non-depleting antibody, however, previous reported anti-VISTA antibodies use IgG1 Fc isotypes that deplete VISTA+ cells by antibody dependent cellular cytotoxicity/complement dependent cytotoxicity and these antibodies have shown fast serum clearance and immune toxicities. Method: Here we used a rational antibody discovery approach to develop the first Fc-independent anti-VISTA antibody, HMBD-002, that binds a computationally predicted functional epitope within the C-C-loop, distinct from other known anti-VISTA antibodies. This epitope is species-conserved allowing robust in vitro and in vivo testing of HMBD-002 in human and murine models of immune activation and cancer including humanized mouse models. Results: We demonstrate here that blockade by HMBD-002 inhibits VISTA binding to potential partners, including V-Set and Immunoglobulin domain containing 3, to reduce myeloid-derived suppression of T cell activity and prevent neutrophil migration. Analysis of immune cell milieu suggests that HMBD-002 treatment stimulates a proinflammatory phenotype characterized by a Th1/Th17 response, recapitulating a phenotype previously noted in VISTA knockout models. This mechanism of action is further supported by immune-competent syngenic and humanized mouse models of colorectal, breast and lung cancer where neutralizing VISTA, without depleting VISTA expressing cells, significantly inhibited tumor growth while decreasing infiltration of suppressive myeloid cells and increasing T cell activity. Finally, we did not observe either the fast serum clearance or immune toxicities that have been reported for IgG1 antibodies. Conclusion: In conclusion, we have shown that VISTA-induced immune suppression can be reversed by blockade of the functional C-C' loop region of VISTA with a first-in-class rationally targeted and non-depleting IgG4 isotype anti-VISTA antibody, HMBD-002. This antibody represents a highly promising novel therapy in the VISTA-suppressed ICT non-responder population. Keywords: immunotherapy; tumor microenvironment.

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