Conditionally Active CD28xVISTA Bispecific Antibodies Promote Myeloid-Driven T-Cell Activation

Abstract Reinvigoration of tumor-reactive T cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T cell–mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by our CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells abundant in most solid tumors. Our lead candidate displayed pH-dependent engagement of VISTA and simultaneous binding to CD28, resulting in VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly binds VISTA+ cells, and co-stimulatory activity was shown in vitro by its ability to activate and expand T cells and enhance T cell–mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. This CD28xVISTA bsAb efficiently inhibited the growth of human VISTA-expressing MC38 tumors in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Our findings support signaling both in cis (between T cell and target cell displaying peptide–MHC complex) and in trans, with stimulation occurring through CD28 clustering outside of the immune synapse. This CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro CRS assays. Thus, our data supports clinical development for solid tumors in combination with anti-PD-1 or TAA-targeted anti-CD3 T-cell engagers.