DataSheet_1_Distinct immune stimulatory effects of anti-human VISTA antibodies are determined by Fc-receptor interaction.pdf

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（PD-1H）是一种免疫调节分子，被视为新一代免疫肿瘤学靶点。VISTA属于免疫球蛋白（Ig）超家族细胞表面分子，主要表达于髓系细胞，在一定程度上也表达于NK细胞与T细胞。既往临床前研究显示，部分靶向VISTA的抗体可传递免疫抑制信号，而另有一些抗体则可触发免疫激活信号。至关重要的是，对于治疗性抗体而言，其同种型骨架可对抗体功能产生显著影响。为阐明免疫激活型抗VISTA抗体的作用机制，我们针对3种不同的抗人VISTA抗体克隆展开研究，每种克隆均搭载目前治疗性抗体常用的3种不同IgG同种型：未修饰IgG1（IgG1-WT）、IgG1-KO（即Fc效应功能减弱的IgG1-LL234,235AA变体）以及IgG4-Pro（即铰链区稳定的IgG4-S228P变体）。我们采用三类模型系统分析抗体功能：以人外周血单个核细胞（peripheral blood mononuclear cells, PBMC）的混合淋巴细胞反应（mixed leukocyte reaction, MLR）作为持续免疫反应的模型，以未刺激的人PBMC作为静息免疫系统的模型，同时使用急性髓系白血病（acute myeloid leukemia, AML）患者样本评估抗VISTA抗体对原代肿瘤组织的作用效果。研究明确了3种抗人VISTA抗体的功能由其IgG同种型骨架决定。健康供者PBMC的混合淋巴细胞反应可被抗VISTA-IgG4-Pro与抗VISTA-IgG1-WT抗体有效增强，具体表现为细胞因子、T细胞激活标志物及T细胞增殖水平升高。然而在单名健康供者的未刺激PBMC培养体系中，仅抗VISTA-IgG1-WT抗体可提升静息髓系细胞表面的激活标志物HLA-DR表达水平及趋化因子水平。值得注意的是，上述两类效应分别需要与不同Fc受体结合：CD64参与增强混合淋巴细胞反应，而CD16则介导静息髓系细胞的激活。此外，抗VISTA-IgG1-KO抗体在所有模型系统中几乎未产生任何影响。同样地，在AML患者样本中，搭载IgG4-Pro骨架的抗VISTA抗体可增强免疫细胞与CD34+ AML癌细胞之间的相互作用（这是一种全新的活性检测指标），而搭载IgG1-KO骨架的抗体则无此效果。综上，拮抗型抗VISTA抗体的免疫激活效应由抗体同种型及与不同Fcγ受体的相互作用共同决定，这凸显了在设计用于免疫肿瘤学应用的免疫激活型抗体治疗药物时，明确此类相互作用的重要性。