Distinct immune stimulatory effects of anti-human VISTA antibodies are determined by Fc-receptor interaction

Abstract

VISTA (PD-1H) is an immune regulatory molecule considered part of the next wave of immuno-oncology targets. VISTA is an immunoglobulin (Ig) superfamily cell surface molecule mainly expressed on myeloid cells, and to some extent on NK cells and T cells. In previous preclinical studies, some VISTA-targeting antibodies provided immune inhibitory signals, while other antibodies triggered immune stimulatory signals. Importantly, for therapeutic antibodies, the isotype backbone can have a strong impact on antibody function. To elucidate the mode of action of immune stimulatory anti-VISTA antibodies, we studied three different anti-human VISTA antibody clones, each on three different IgG isotypes currently used for therapeutic antibodies: unaltered IgG1 (IgG1-WT), IgG1-KO (IgG1-LL234,235AA-variant with reduced Fc-effector function), and IgG4-Pro (IgG4- S228P-variant with stabilized hinge region). Antibody functionality was analysed in mixed leukocyte reaction (MLR) of human peripheral blood mononuclear cells (PBMCs), as a model system for ongoing immune reactions, on unstimulated human PBMCs, as a model system for a resting immune system, and also on acute myeloid leukemia (AML) patient samples to evaluate anti-VISTA antibody effects on primary tumor material. The functions of three anti-human VISTA antibodies were determined by their IgG isotype backbones. An MLR of healthy donor PBMCs was effectively augmented by anti-VISTA-IgG4-Pro and anti-VISTA-IgG1-WT antibodies, as indicated by increased levels of cytokines, T cell activation markers and T cell proliferation. However, in a culture of unstimulated PBMCs of single healthy donors, only anti-VISTA-IgG1-WT antibodies increased the activation marker HLA-DR on resting myeloid cells, and chemokine levels. Interestingly, interactions with different Fc-receptors were required for these effects, namely CD64 for augmentation of MLR, and CD16 for activation of resting myeloid cells. Furthermore, anti-VISTA-IgG1-KO antibodies had nearly no impact in any model system. Similarly, in AML patient samples, anti-VISTA-antibody on IgG4-Pro backbone, but not on IgG1-KO backbone, increased interactions, as a novel readout of activity, between immune cells and CD34+ AML cancer cells. In conclusion, the immune stimulatory effects of antagonistic anti-VISTA antibodies are defined by the antibody isotype and interaction with different Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for immuno-oncology applications.

VISTA expression and patient selection for immune-based anticancer therapy

“…In particular, the broad expression of VISTA on myeloid cells, including MDSCs, may lead to systemic consequences for inhibiting VISTA that extend beyond the TME. Cytokine release is another concern which has been demonstrated in a mixed leukocyte reaction (MLR) of human peripheral blood mononuclear cells (PBMCs) with multiple anti-VISTA monoclonal antibodies which is dependent on them having functional Fc domains ( 90 ). Cytokine release with peak levels of cytokines between 2-6 hrs has been seen with Fc-active-anti-VISTA blockade in a human early phase trial which investigators will need to mitigate through dosing adjustments and additional anti-inflammatory medications ( 76 ).…”

Conditionally Active, pH-Sensitive Immunoregulatory Antibodies Targeting VISTA and CTLA-4 Lead an Emerging Class of Cancer Therapeutics

“…At subtherapeutic dose levels (0.3 mgs/kg IV), grade 3 cytokine release syndrome and encephalopathy were encountered, leading JNJ to stop the trial [ 18 ]. JNJ-61610588 utilized an Fc-competent IgG1 framework, which was chosen based on preclinical studies showing that Fc-incompetent frameworks were significantly inferior in terms of immunostimulatory and anti-tumor effects [ 19 , 20 ]. Although not formally demonstrated with JNJ-61610588, based on in vitro studies showing IgG1-dependent myeloid activation, it appears likely that the coating of myeloid cells in the blood with an IgG1 anti-VISTA mAb led to activation in circulation, resulting in cytokine release syndrome (CRS).…”