Boshuizen Ronald S, Marsden Catherine, Turkstra Johan, Rossant Christine J, Slootstra Jerry, Copley Clive, Schwamborn Klaus
a Pepscan Therapeutics B.V. ; Lelystad , The Netherlands.
MAbs. 2014;6(6):1415-24. doi: 10.4161/mabs.36237.
Development of functional monoclonal antibodies against intractable GPCR targets.
Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.
The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.
The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.
The CXC chemokine receptor-2 (CXCR2) is a member of the large 'family A' of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor. The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.
针对难治性G蛋白偶联受体（GPCR）靶点开发功能性单克隆抗体。
鉴定出模拟配体结合位点的结构化肽，将其用于淘选以富集结合剂群体，随后用过度表达受体的细胞对该群体进行筛选，从而获得功能性单克隆抗体。
这些技术的组合为在相对小规模的研究中开发针对CXCR2的功能性单克隆抗体提供了一种成功的策略方法。
所展示的技术组合可能适用于其他 notoriously difficult 的GPCR靶点。
CXC趋化因子受体2（CXCR2）是G蛋白偶联受体大家族“A类”的成员，在多种类型的癌细胞中过度表达。CXCR2可被多种配体激活，包括白细胞介素8（IL-8）和生长相关蛋白α（Gro-α）。因此，能够阻断配体 - 受体相互作用的单克隆抗体具有治疗意义；然而，针对高度结构化的GPCR蛋白开发具有生物活性的抗体具有挑战性。在此，我们展示了一组技术组合，可改进针对天然CXCR2受体的功能性单克隆抗体的发现。通过用IL-8配体筛选肽库鉴定出CXCR2的IL-8结合位点，然后将其重建为可溶性合成肽。这些肽用作抗原，探测抗体片段噬菌体展示文库，以获得与CXCR2的IL-8结合位点结合的亚群。通过以过度表达CXCR2的细胞作为不同抗原源进行额外一轮筛选，进一步富集噬菌体群体。对来自CXCR2特异性噬菌体克隆的单链抗体（scFv）进行测序，并转化为单克隆抗体。所获得的抗体特异性结合表达CXCR2的细胞，并抑制IL-8和Gro-α诱导的β - 抑制蛋白募集，IC50值分别为0.3和0.2 nM，并且比用相同肽抗原通过经典杂交瘤技术获得的鼠单克隆抗体（分别为18和19 nM）效力显著更高。根据表位作图研究，抗体效力在很大程度上由包含IL-8和Gro-α结合位点的N端表位决定。所展示的包括使用不同抗原源的体外技术的策略组合，是通过经典杂交瘤技术开发功能性单克隆抗体的有力替代方法，并且可能适用于其他GPCR靶点。
