VISTA, an inhibitory myeloid-T-cell checkpoint, holds promise as a target for cancer immunotherapy. However, its effective targeting has been impeded by issues such as rapid clearance and cytokine release syndrome observed with previous VISTA antibodies. Here we demonstrate that SNS-101, a newly developed pH-selective VISTA antibody, addresses these challenges. Structural and biochemical analyses confirmed the pH-selectivity and unique epitope targeted by SNS-101. These properties confer favorable pharmacokinetic and safety profiles on SNS-101. In syngeneic tumor models utilizing human VISTA knock-in mice, SNS-101 shows in vivo efficacy when combined with a PD-1 inhibitor, modulates cytokine and chemokine signaling, and alters the tumor microenvironment. In summary, SNS-101, currently in Phase I clinical trials, emerges as a promising therapeutic biologic for a wide range of patients whose cancer is refractory to current immunotherapy regimens.

Immune checkpoints and other immunoregulatory targets can be difficult to precisely target due to expression on non-tumor immune cells critical to maintaining immune homeostasis in healthy tissues. On-target/off-tumor binding of therapeutics results in significant pharmacokinetic and pharmacodynamic problems. Target-mediated drug disposition (TMDD) significantly limits effective intratumoral drug levels and adversely affects anti-tumor efficacy. Target engagement outside the tumor environment may lead to severe immune-related adverse events (irAEs), resulting in a narrowing of the therapeutic window, sub-optimal dosing, or cessation of drug development altogether. Overcoming these challenges has become tractable through recent advances in antibody engineering and screening approaches. Here, we review the discovery and development of conditionally active antibodies with minimal binding to target at physiologic pH but high-affinity target binding at the low pH of the tumor microenvironment by focusing on the discovery and improved properties of pH-dependent mAbs targeting two T cell checkpoints, VISTA and CTLA-4.

Reinvigoration of tumor-reactive T cells using costimulatory bispecific antibodies (bsAb) 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 CD28xV-domain Ig-containing suppressor of T-cell activation (VISTA) bsAbs to act specifically within the acidic tumor microenvironment, aiming for enhanced T cell–mediated cancer cell killing while minimizing systemic T-cell activation and cytokine release syndrome 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 costimulatory activity was shown in vitro by its ability to activate and expand T cells and enhance T cell–mediated cancer cell killing in cocultures of human peripheral blood mononuclear cells and cancer cells in the presence of a tumor-associated antigen–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 cytokine release syndrome assays. Thus, our data support clinical development for solid tumors in combination with anti–PD-1 or tumor-associated antigen–targeted anti-CD3 T-cell engagers.

Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary 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 CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects. We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation 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. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors.

Tumor Growth Inhibition Summary and Statistics