Highlights d The crystal structure of the human VISTA extracellular domain was determined d VISTA contains two unique disulfides and an extended C-C 0 loop d The epitope of an inhibitor antibody was mapped to a threeresidue surface d The antibody epitope was found to overlap with the VISTA-VSIG3 binding interface

V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3 proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.

Adoptive cell therapy is a rapidly advancing approach to cancer immunotherapy that seeks to facilitate antitumor responses by introducing potent effector cells into the tumor microenvironment. Expanded autologous T cells, particularly T cells with engineered T cell receptors (TCR) and chimeric antigen receptor-T cells have had success in various hematologic malignancies but have faced challenges when applied to solid tumors. As a result, other immune subpopulations may provide valuable and orthogonal options for treatment. Natural killer (NK) cells offer the possibility of significant tumor clearance and recruitment of additional immune subpopulations without the need for prior antigen presentation like in T or B cells that could require removal of endogenous antigen specificity mediated via the T cell receptor (TCR and/or the B ecll receptor (BCR). In recent years, NK cells have been demonstrated to be increasingly important players in the immune response against cancer. Here, we review multiple avenues for allogeneic NK cell therapy, including derivation of NK cells from peripheral blood or umbilical cord blood, the NK-92 immortalized cell line, and induced pluripotent stem cells (iPSCs). We also describe the potential of engineering iPSC-derived NK cells and the utility of this platform. Finally, we consider the benefits and drawbacks of each approach and discuss recent developments in the manufacturing and genetic or metabolic engineering of NK cells to have robust and prolonged antitumor responses in preclinical and clinical settings.

V-domain Ig Suppressor of T cell Activation (VISTA) is an immune checkpoint protein that inhibits the Tcell response against cancer. Similar to PD-1 and CTLA-4, antibodies that block VISTA signaling can release the brakes of the immune system and promote tumor clearance. VISTA has an Ig-like fold, but little is known about its structure and mechanism of action. Here, we report a 1.85 Å crystal structure of the human VISTA extracellular domain and highlight structural features that make VISTA unique among B7 family members.Through fine-epitope mapping, we also identify solvent-exposed residues that underlie binding to a clinically relevant anti-VISTA antibody. This antibody-binding region is also shown to interact with V-set and Ig domaincontaining 3 (VSIG3), the recently proposed functional binding partner of VISTA. The structure and functional epitope determined here will help guide future drug development efforts against this important checkpoint target.

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