Directing selective complement activation towards tumour cells is an attractive strategy to promote their elimination. In the present work, we have generated heteromultimeric immunoconjugates that selectively activate the complement alternative pathway (AP) on tumour cells. We used the C4b-binding protein C-terminal-α-/β-chain scaffold for multimerisation to generate heteromultimeric immunoconjugates displaying (a) a multivalent-positive regulator of the AP, the human factor H-related protein 4 (FHR4) with; (b) a multivalent targeting function directed against erbB2 (HER2); and (c) a monovalent enhanced GFP tracking function. Two distinct VH H targeting two different epitopes against HER2 and competing either with trastuzumab or with pertuzumab-recognising epitopes [VH H(T) or VH H(P)], respectively, were used as HER2 anchoring moieties. Optimised high-FHR4 valence heteromultimeric immunoconjugates [FHR4/VH H(T) or FHR4/VH H(P)] were selected by sequential cell cloning and a selective multistep His-Trap purification. Optimised FHR4-heteromultimeric immunoconjugates successfully overcame FH-mediated complement inhibition threshold, causing increased C3b deposition on SK-OV-3, BT474 and SK-BR3 tumour cells, and increased formation of lytic membrane attack complex densities and complement-dependent cytotoxicity (CDC). CDC varies according to the pattern expression and densities of membrane-anchored complement regulatory proteins on tumour cell surfaces. In addition, opsonised BT474 tumour cells were efficiently phagocytosed by macrophages through complement-dependent cell-mediated cytotoxicity. We showed that the degree of FHR4-multivalency within the multimeric immunoconjugates was the key element to efficiently compete and deregulate FH and FH-mediated convertase decay locally on tumour cell surface. FHR4 can thus represent a novel therapeutic molecule, when expressed as a multimeric entity and associated with an anchoring system, to locally shift the complement steady-state towards activation on tumour cell surface.

中文翻译：

---

基于FHR4的免疫缀合物将补体依赖性细胞毒性和吞噬作用导向HER2阳性癌细胞。

将选择性补体激活导向肿瘤细胞是促进其消除的有吸引力的策略。在目前的工作中，我们已经产生了异源多聚体免疫偶联物，其选择性激活肿瘤细胞上的补体旁路途径（AP）。我们使用了C4b结合蛋白C-末端-α-/β-链支架进行多聚化，以产生异源多聚体免疫结合物，显示出（a）AP的多价阳性调节剂，与人因子H相关蛋白4（FHR4）结合；（b）针对erbB2（HER2）的多价靶向功能；（c）单价增强的GFP追踪功能。将针对HER2的两个不同表位并与曲妥珠单抗或与帕妥珠单抗识别的表位[VH H（T）或VH H（P）]竞争的两个不同的VH H分别用作HER2锚定部分。通过顺序细胞克隆和选择性多步His-Trap纯化，选择了优化的高FHR4价异多聚体免疫偶联物[FHR4 / VH H（T）或FHR4 / VH H（P）]。优化的FHR4-异多聚体免疫偶联物成功克服了FH介导的补体抑制阈值，导致C3b在SK-OV-3，BT474和SK-BR3肿瘤细胞上的沉积增加，并且裂解膜攻击复合物密度和补体依赖性细胞毒性（CDC）的形成增加。CDC根据肿瘤细胞表面上膜锚定的补体调节蛋白的模式表达和密度而变化。此外，调理剂BT474肿瘤细胞通过补体依赖性细胞介导的细胞毒性被巨噬细胞有效吞噬。我们表明，多聚体免疫结合物中FHR4多价的程度是在肿瘤细胞表面局部有效竞争和解除FH和FH介导的转化酶衰减的关键因素。因此，当表达为多聚体实体并与锚定系统关联时，FHR4可以代表一种新型治疗分子，可将补体稳态局部转移至肿瘤细胞表面上的激活状态。