Abstract 1548: Acidic pH selective binding of VISTA to PSGL-1 and anti-tumor activity of combined VISTA and PD-1 blockade

Background: Therapeutic blockade of the immune checkpoints CTLA-4 and PD-1/PD-L1 has provided durable survival benefits in multiple malignancies. However, additional treatment options are often required to maximally reverse immune dysfunction. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an orphan B7 family ligand that is highly expressed on immunosuppressive myeloid cells and has been shown to inhibit T-cell responses in vitro and in preclinical models of cancer. Here we report that VISTA, a ligand for the receptor P-selectin glycoprotein ligand-1 (PSGL-1), uses a histidine-rich interface to engage PSGL-1 and suppress immune responses selectively in acidic environments, such as tumor beds. Methods: Recombinant VISTA multimers were used to assess binding to cells and recombinant PSGL-1 over a range of pH values (6.0-7.4). Antibodies against human and mouse VISTA were used to map binding and functional epitopes. Acidic pH receptor-based ligand capture was used to identify PSGL-1 as a VISTA receptor. X-ray crystallography was used to resolve the VISTA structure in complex with an anti-VISTA antigen-binding fragment. The MC38 mouse tumor model was used to assess the effects of VISTA deficiency and the effects of VISTA antibody blockade alone and combined with anti-PD-1 in vivo. Results: Recombinant VISTA bound leukocytes at pH 6.0 but was not detectable at pH 7.4. Antibodies in a single epitope bin blocked VISTA binding and reversed VISTA suppression of T cells. VISTA-mediated inhibition of T cells was detectable at pH 7.4 but was more pronounced below pH 7.0, suggesting that VISTA functions selectively in acidic conditions. VISTA’s structure was resolved at 1.6 Å and characterized by a histidine-rich extension of the immunoglobulin V domain central β-sheet. VISTA blocking antibodies, but not nonblocking antibodies, bound this β-sheet region. Engineered antibodies could distinguish this epitope in its active and inactive states at acidic and neutral pH, respectively. Receptor capture on T cells at acidic pH identified PSGL-1 as a VISTA receptor. T-cell PSGL-1 CRISPR ablated VISTA binding, whereas PSGL-1 expression on CHO cells conferred VISTA binding at acidic pH. Thus, an antibody that blocks mouse VISTA binding to mouse T cells at acidic pH combined with a PD-1 blocking antibody was shown to enhance anti-tumor T-cell responses and drive MC38 tumor rejection in vivo. Conclusions: VISTA is a highly pH-selective ligand for PSGL-1. VISTA a