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首页> 外文期刊>BMC Biotechnology >Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages
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Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages

机译：靶向针对表达唾液酸粘附素的巨噬细胞的肽和蛋白质的重组抗体的开发

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Background Sialoadhesin (Sn)-expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. This makes Sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. Furthermore, since Sn was shown to be an endocytic receptor, the conjugation of effector molecules to an Sn-specific ligand should allow intracellular delivery of these conjugates. Previously, we developed functional Sn-specific immunoconjugates that were generated via chemical coupling. Although successful, the system requires significant optimization for each immunoconjugate to be made. To generate a more flexible and controlled system, we developed a recombinant antibody vector allowing the creation of genetic antibody fusion constructs. This paper reports on the characterization of the recombinant antibody and the evaluation of its use for Sn-directed targeting. Results The variable domains of the porcine Sn-specific monoclonal antibody 41D3 were sequenced and cloned in frame with a mouse IgG1 backbone. Transfection of HEK293T cells with the resulting plasmid led to the secretion of fully assembled IgG into the culture medium. This recombinant antibody rec41D3 was shown to specifically bind to porcine Sn with a comparable affinity as the native monoclonal antibody. In addition, rec41D3 also induced Sn endocytosis in primary macrophages and resided for prolonged times in early/late endosomes. To allow the generation of antibody fusion constructs, a multiple cloning site was introduced at the C-terminus of the heavy chain. Two fusion constructs were generated, one containing a V5 peptide tag and one containing an eGFP molecule. Both constructs were shown to be efficiently produced in HEK293T cells and easily purified using standard protein G chromatography. In addition, both V5 and eGFP were shown to be co-internalized together with rec41D3 into Sn-expressing primary macrophages. Conclusions A recombinant antibody allowing targeted delivery of peptides and proteins to Sn-expressing macrophages was developed. Production and purification of antibody fusion constructs was possible without major optimization and with batch to batch consistency, confirming the development of a versatile antibody vector to evaluate Sn-directed targeting strategies in a porcine animal model.
机译：背景表达Sialoadhesin（Sn）的单核细胞/巨噬细胞已与多种疾病（如炎性和自身免疫性疾病以及病毒感染）相关，并且它们似乎在适应性免疫反应的启动中也起作用。这使得表达Sn的细胞不仅对细胞定向治疗具有吸引力，而且对疫苗接种也具有吸引力。此外，由于显示出Sn是胞吞受体，因此效应子分子与Sn特异性配体的缀合应允许这些缀合物的细胞内递送。以前，我们开发了通过化学偶联生成的功能性Sn特异性免疫偶联物。尽管成功，但该系统需要对要制备的每种免疫偶联物进行重大优化。为了产生更灵活和受控的系统，我们开发了重组抗体载体，可创建遗传抗体融合构建体。本文报道了重组抗体的表征及其在Sn定向靶向中的应用评估。结果对猪Sn特异性单克隆抗体41D3的可变域进行了测序，并与小鼠IgG1骨架进行了框内克隆。用所得质粒转染HEK293T细胞导致完全组装的IgG分泌到培养基中。该重组抗体rec41D3显示出以与天然单克隆抗体相当的亲和力特异性结合猪Sn。此外，rec41D3还诱导初级巨噬细胞中的Sn内吞作用，并在早期/晚期内体中长时间停留。为了产生抗体融合构建体，在重链的C末端引入了多个克隆位点。产生了两个融合构建体，一个包含V5肽标签，另一个包含eGFP分子。两种构建体均显示可在HEK293T细胞中有效产生，并易于使用标准蛋白质G色谱法纯化。此外，显示V5和eGFP与rec41D3一起共内在表达Sn的初级巨噬细胞中。结论开发了一种重组抗体，可将肽段和蛋白质靶向递送至表达Sn的巨噬细胞。抗体融合构建体的生产和纯化无需进行重大优化即可实现，而且批次之间具有一致性，从而证实了开发多功能抗体载体以评估猪动物模型中Sn定向靶向策略的可行性。

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