Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.
Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.
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DOI：
10.1042/bcj20200674
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发表时间：
2021-01-15
期刊：
影响因子：
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通讯作者：
Hupp TR
中科院分区：
文献类型：
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作者：
Uhrik L;Hernychova L;Muller P;Kalathiya U;Lisowska MM;Kocikowski M;Parys M;Faktor J;Nekulova M;Nortcliffe C;Zatloukalova P;Ruetgen B;Fahraeus R;Ball KL;Argyle DJ;Vojtesek B;Hupp TR
通过产生产生对犬CD 20具有特异性的鼠单克隆抗体的杂交瘤细胞，正在B细胞淋巴瘤中开发比较犬-人治疗模型。杂交瘤细胞产生两条轻链，轻链-3和轻链-7。然而，任一轻链对真实全长杂交瘤衍生IgG的贡献是不确定的。使用质谱法鉴定两条轻链中仅一条轻链-7在全长IgG中占优势。基因合成产生重组鼠-犬嵌合单克隆抗体，其表达重组IgG与CD 20结合的轻链-7。使用轻链-7作为参考序列，使用氢氘交换质谱法来鉴定与CD 20抗原结合有关的主要CDR区。在氘化反应的早期，CD 20抗原抑制了CDR 3（VH）的氘化。在稍后的时间点，氘抑制发生在CDR 2（VH）和CDR 2（VL）处，同时维持CDR 3（VH）相互作用。这些数据表明，CDR 3（VH）作为主要的抗原对接基序起作用，并且在抗原结合后的稍后时间点诱导抗体聚集。这些方法定义了使用嵌套酶促反应和氢氘交换质谱法对CDR接触进行精细定位的方法。这些数据支持进一步开发一种工程化的、合成的犬-鼠单克隆抗体，集中在CDR 3（VH）上，用作模拟人-鼠嵌合抗CD 20抗体利妥昔单抗的犬淋巴瘤治疗剂。
A comparative canine–human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine–canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine–murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human–murine chimeric anti-CD20 antibody Rituximab.