Silkina Marina Vladimirovna, Kartseva Alena Sergeevna, Riabko Alena Konstantinovna, Makarova Mariia Aleksandrovna, Rogozin Metkhun Madibronovich, Romanenko Yana Olegovna, Shemyakin Igor Georgievich, Dyatlov Ivan Alekseevich, Firstova Victoria Valerievna
State Research Center for Applied Microbiology and Biotechnology (SRCAMB), Obolensk 142279, Russia.
Bioimpacts. 2024;14(4):27680. doi: 10.34172/bi.2023.27680. Epub 2023 Dec 30.
Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies.
Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay.
We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity .
The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure.
肉毒杆菌神经毒素（BoNTs）可导致肉毒中毒，是已知最有效的天然毒素。用中和单克隆抗体（MAbs）进行免疫治疗被认为是对BoNT暴露最有效的即时反应。杂交瘤技术仍然是生产具有天然配对免疫球蛋白基因且保留免疫细胞固有功能的单克隆抗体的首选方法。亲和力成熟的人抗体库可能是抗BoNTs抗体治疗药物的理想来源。为了开发新型A型肉毒杆菌神经毒素（BoNT/A）免疫治疗药物，由于要获得产生天然抗体的杂交瘤，可使用通过流式细胞术分选的来自反复注射BoNT/A用于美容肉毒杆菌治疗的供体的浆母细胞和活化记忆B细胞。
在注射BoNT/A后7天采集的全血中分离浆母细胞和活化记忆B细胞，并通过流式细胞术进行分选。然后将分选的细胞与K6H6/B5细胞系进行电融合，从而产生天然人单克隆抗体（huMAbs）的产生细胞。然后通过亲和层析纯化获得的3种抗体，通过蛋白质印迹分析其结合情况，并通过荧光共振能量转移分析其中和作用。
我们成功创建了3个杂交瘤，它们分泌针对天然BoNT/A和BoNT/A蛋白水解结构域（轻链，LC）的特异性huMAbs。1B9抗体还直接抑制BoNT/A的催化活性。
使用在免疫反应高峰期（免疫发生第7天）分离的活化浆母细胞和记忆B细胞进行杂交，这些细胞尚未完成分化的终末阶段，但已经历体细胞超突变，这使我们即使在供体免疫反应较弱（血液中特异性抗体和特异性B细胞水平较低）时也能获得特异性huMAbs。一种BoNT/A LC特异性抗体能够通过以前与中和BoNT的抗体无关的机制有效抑制BoNT/A。针对BoNT LC的特异性抗体可能是抗BoNT暴露抗体混合物的有价值成分。
