Antibodies that target a clinically relevant group of receptors within the tumor necrosis factor receptor superfamily (TNFRSF), including CD40 and CD95 (Fas/Apo-1), also require binding to Fc gamma receptors (FcγRs) to elicit a strong agonistic activity. This FcγR dependency largely relies on the mere cellular anchoring through the antibody’s Fc domain and does not involve the engagement of FcγR signaling. The aim of this study was to elicit agonistic activity from αCD40 and αCD95 antibodies in a myeloma cell anchoring-controlled FcγR-independent manner. For this purpose, various antibody variants (IgG1, IgG1_N297A, Fab₂) against the TNFRSF members CD40 and CD95 were genetically fused to a single-chain-encoded B-cell activating factor (scBaff) trimer as a C-terminal myeloma-specific anchoring domain substituting for Fc domain-mediated FcγR binding. The antibody-scBaff fusion proteins were evaluated in binding studies and functional assays using tumor cell lines expressing one or more of the three receptors of Baff: BaffR, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA). Cellular binding studies showed that the binding properties of the different domains within the fusion proteins remained fully intact in the antibody-scBaff fusion proteins. In co-culture assays of CD40- and CD95-responsive cells with BaffR, BCMA or TACI expressing anchoring cells, the antibody fusion proteins displayed strong agonism while only minor receptor stimulation was observed in co-cultures with cells without expression of Baff-interacting receptors. Thus, our CD40 and CD95 antibody fusion proteins display myeloma cell-dependent activity and promise reduced systemic side effects compared to conventional CD40 and CD95 agonists.

靶向肿瘤坏死因子受体超家族（TNFRSF）中临床相关受体组的抗体，包括CD40和CD95（Fas / Apo-1），也需要与Fcγ受体（FcγRs）结合以引起强烈的激动活性。FcγR依赖性在很大程度上取决于通过抗体Fc结构域的单纯细胞锚定，并且不涉及FcγR信号传导的参与。这项研究的目的是以骨髓瘤细胞锚定控制的FcγR独立方式从αCD40和αCD95抗体中激发激动活性。为此目的，各种抗体变体（IgG1，IgG1 _N297A，Fab ₂）针对TNFRSF的成员，将CD40和CD95遗传融合到单链编码的B细胞活化因子（scBaff）三聚体上，作为C端骨髓瘤特异性锚定结构域取代Fc结构域介导的FcγR结合。使用表达Baff三种受体中的一种或多种的肿瘤细胞系，在结合研究和功能测定中评估了抗体-scBaff融合蛋白：BaffR，跨膜激活物和CAML相互作用物（TACI）和B细胞成熟抗原（BCMA）。细胞结合研究表明，融合蛋白内不同结构域的结合特性在抗体-scBaff融合蛋白中仍保持完整。在CD40和CD95响应细胞与表达BaffR，BCMA或TACI的锚定细胞的共培养试验中，抗体融合蛋白表现出强烈的激动作用，而在与不表达Baff相互作用受体的细胞共培养中仅观察到很小的受体刺激。因此，与传统的CD40和CD95激动剂相比，我们的CD40和CD95抗体融合蛋白显示出骨髓瘤细胞依赖性活性，并有望减少全身性副作用。