Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.
Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.
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DOI：
10.4161/19420862.2014.985158
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发表时间：
2015
期刊：
影响因子：
5.3
通讯作者：
Vaughan TJ
中科院分区：
文献类型：
--
作者：
Douthwaite JA;Sridharan S;Huntington C;Hammersley J;Marwood R;Hakulinen JK;Ek M;Sjögren T;Rider D;Privezentzev C;Seaman JC;Cariuk P;Knights V;Young J;Wilkinson T;Sleeman M;Finch DK;Lowe DC;Vaughan TJ
靶向 G 蛋白偶联受体 (GPCR) 的治疗性单克隆抗体对于干预多种疾病过程是理想的。由于许多 GPCR 作为纯化蛋白缺乏稳定性，因此发现此类抗体具有挑战性。我们在这里描述了 Fpro0165 的产生，这是一种人抗甲酰基肽受体 1 (FPR1) 抗体，通过对表达人可变区基因的转基因小鼠进行免疫而衍生的抗体的可变域工程产生。使用 HEK 细胞上表达的 FPR1 进行免疫和选择，以及用于抗体筛选的钙释放细胞测定，实现了抗体分离和随后使用噬菌体展示的亲和力、效力和物种交叉反应性工程。 Fpro0165 显示完全中和甲酰肽介导的原代人中性粒细胞的激活。 Fpro0165 Fab 的晶体结构显示出一个由 24 个氨基酸组成的长而突出的 VH CDR3，并且与 FPR1 的同源模型进行计算机对接表明，这个长的 VH CDR3 对于抗体的预测结合模式至关重要。抗体突变研究确定长 VH CDR3 的顶点是介导抗体物种交叉反应性的关键。这项研究阐述了一种针对典型棘手的膜蛋白（例如 GPCR）进行抗体发现和亲和工程的方法。
Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.