Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion
Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion
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DOI：
10.1016/j.jmb.2010.04.001
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发表时间：
2010-06-11
影响因子：
5.6
通讯作者：
Bonvini, Ezio
中科院分区：
文献类型：
--
作者：
Johnson, Syd;Burke, Stephen;Bonvini, Ezio
能够重定向免疫效应细胞的裂解潜力以杀死肿瘤靶标的双特异性抗体长期以来被认为是潜在有效的生物治疗干预。不幸的是，由于生产效率低和稳定性差，生产这种分子的努力受到限制。在这里，我们描述了一种基于共价连接的双特异性双抗体结构的新型Fv衍生策略，我们称之为双亲和力重靶向（DART）。作为模型系统，我们将效应细胞上对人CD16（Fc γ RIII）特异性的Fv连接至对小鼠或人CD32B（Fc γ RIIB）（正常B细胞和肿瘤靶抗原）特异性的Fv。DART蛋白在哺乳动物细胞中以高水平产生，保留各自亲本Fv结构域的结合活性以及双特异性结合，并且显示延长的储存和血清稳定性。在功能上，DART分子在针对B淋巴瘤细胞系重新靶向人PBMC以及介导培养物中的自体B细胞耗竭方面表现出极其有效的剂量依赖性细胞毒性。小鼠体内研究表明，有效的B细胞耗竭依赖于效应细胞上的CD16 A和B细胞靶标上的CD32 B的转基因表达。此外，DART蛋白在人伯基特淋巴瘤细胞异种移植模型中显示出有效的体内保护活性。因此，DART代表了一种生物学上有效的形式，其提供了一种通用的平台，用于产生用于重定向杀伤的双特异性抗体片段，并且通过选择适当的结合配偶体，用于肿瘤细胞毒性之外的应用。(C)2010爱思唯尔有限公司保留所有权利。
Bispecific antibodies capable of redirecting the lyric potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (Fc gamma RIII) on effector cells to an Fv specific for mouse or human CD32B (Fc gamma RIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity. (C) 2010 Elsevier Ltd. All rights reserved.