DataSheet_1_Enhancement of complement-dependent cytotoxicity by linking factor-H derived short consensus repeats 19-20 to CD20 antibodies.pdf

Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19–20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.

抗体介导的补体依赖细胞毒性（complement-dependent cytotoxicity, CDC）对恶性细胞的杀伤效应受多种补体调控蛋白调控，其中包括抑制性补体因子H（factor H, fH）。fH由20个短共有重复序列（short consensus repeat, SCR）组成，具备特定的功能结构域。既往研究表明，来源于fH的SCR19-20片段（SCR1920）可在慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）细胞表面置换全长fH，从而使CLL细胞对靶向CD20的治疗性单克隆抗体（monoclonal antibody, mAb）诱导的CDC更为敏感。
为此，本研究构建了慢病毒载体，以三款临床获批的亲本单克隆抗体——利妥昔单抗、奥妥珠单抗与奥法妥木单抗为基础，分别制备可稳定表达mAb-SCR融合变体的细胞系。流式细胞术检测结果显示，经SCR结构域修饰的单克隆抗体并未削弱其与CD20的结合能力。与亲本单克隆抗体相比，这类融合抗体的体外裂解活性显著提升：实验证实，相较于亲本抗体，融合抗体可通过CDC实现特异性且剂量依赖性的靶细胞清除。
进一步实验发现，自然杀伤细胞（natural killer cell, NK细胞）的耗竭并不会影响对CLL细胞的裂解，提示抗体依赖的细胞介导的细胞毒性作用（antibody-dependent cellular cytotoxicity, ADCC）在该体系中发挥的作用较为有限。综上，本研究明确了CDC在单克隆抗体清除CLL细胞过程中的关键作用，并提出了一种通过将fH来源的SCR1920与单克隆抗体直接融合以增强CDC的全新策略。