Klose Diana, Woitok Mira, Niesen Judith, Beerli Roger R, Grawunder Ulf, Fischer Rainer, Barth Stefan, Fendel Rolf, Nachreiner Thomas
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
Department of Experimental Medicine and Immunotherapy, Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Aachen, Germany.
PLoS One. 2017 Jul 13;12(7):e0180305. doi: 10.1371/journal.pone.0180305. eCollection 2017.
The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb™ technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells.
通过自身反应性B细胞独特的B细胞受体（BCR）进行抗原特异性靶向，是体液免疫全身性抑制的一种新颖且有前景的替代方法。我们基于现有的免疫毒素生成并表征了溶细胞融合蛋白，该免疫毒素包含破伤风类毒素片段C（TTC）作为靶向成分，以及修饰的铜绿假单胞菌外毒素A（ETA'）作为细胞毒性成分。对该免疫毒素进行重新构建，用粒酶B突变体R201K或微管相关蛋白Tau（MAPTau）作为人类效应域取代ETA'。利用Transpo-mAb™技术开发的重组人淋巴细胞系对新型溶细胞融合蛋白进行了表征。编码嵌合型TTC反应性免疫球蛋白G的基因成功整合到前体B细胞系REH的基因组中，使得这些细胞能够在其表面呈现TTC反应性BCR。这些细胞被用于研究GrB（R201K）-TTC和TTC-MAPTau的特异性细胞毒性，结果显示丝氨酸蛋白酶抑制剂9抗性的粒酶B R201K突变体在淋巴细胞系中特异性诱导细胞凋亡。我们的数据证实，含有粒酶B（R201K）的基于抗原的融合蛋白是清除自身反应性B细胞的合适候选物。
