Steidl Stefan, Ratsch Olaf, Brocks Bodo, Dürr Manuela, Thomassen-Wolf Elisabeth
MorphoSys AG, Lena-Christ-Str. 48, Martinsried 82152, Germany.
Mol Immunol. 2008 Nov;46(1):135-44. doi: 10.1016/j.molimm.2008.07.013. Epub 2008 Aug 21.
The mammalian immune system applies somatic hypermutation to select for antibodies with improved dissociation rates in vivo up to an intrinsic limit, previously termed as affinity ceiling. However, for certain therapeutic applications it may be desirable to further improve antibody affinities beyond that limit. In this study the selection of antibodies specific for the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) from the HuCAL GOLD human antibody library is described. In order to increase affinity and also functional activity, in vitro affinity maturation of a pool of lead Fab candidates was carried out. CDR-L3 and parallel CDR-H2 diversification using trinucleotide consensus cassettes were followed by the combination of optimized CDR-L3 and CDR-H2 leading to a 5000-fold improved affinity finally reaching a K(D) of 400 fM. Cytokine neutralizing potential of MOR04357 was evaluated in a TF-1 proliferation assay. Along with affinity optimization a 2000-fold increase in potency was observed compared to the parental antibody. Due to species cross-reactivity MOR04357 also blocks rat GM-CSF induced proliferation of FDCP-1 cells. Receptor inhibition studies showed that MOR04357 prevents the interaction of GM-CSF with the GM-CSF receptor alpha chain. As a consequence this leads to a blockade in signal transduction as measured by abolished STAT5 phosphorylation in the presence of GM-CSF and antibody. Due to its pro-inflammatory role GM-CSF has been implicated in the pathophysiology of inflammatory diseases like rheumatoid arthritis or asthma. Based on the mode of action described herein MOR04357 shows favourable antibody features as a potential drug candidate.
哺乳动物免疫系统通过体细胞超突变在体内选择解离速率得到改善的抗体，直至达到一个内在极限，该极限先前被称为亲和力上限。然而，对于某些治疗应用而言，可能希望进一步提高抗体亲和力，使其超过该极限。在本研究中，描述了从HuCAL GOLD人源抗体库中筛选针对促炎细胞因子粒细胞-巨噬细胞集落刺激因子（GM-CSF）的抗体。为了提高亲和力以及功能活性，对一组先导Fab候选物进行了体外亲和力成熟。使用三核苷酸共有盒对互补决定区3（CDR-L3）和平行互补决定区2（CDR-H2）进行多样化，随后将优化后的CDR-L3和CDR-H2组合，最终使亲和力提高了5000倍，达到400 fM的解离常数（K(D)）。在TF-1增殖试验中评估了MOR04357的细胞因子中和潜力。与亲本抗体相比，在优化亲和力的同时，效力提高了2000倍。由于种间交叉反应性，MOR04357也能阻断大鼠GM-CSF诱导的FDCP-1细胞增殖。受体抑制研究表明，MOR04357可阻止GM-CSF与GM-CSF受体α链相互作用。因此，这导致信号转导受阻，如在存在GM-CSF和抗体的情况下，信号转导及转录激活因子5（STAT5）磷酸化被消除所测量的那样。由于其促炎作用，GM-CSF与类风湿性关节炎或哮喘等炎症性疾病的病理生理学有关。基于本文所述的作用方式，MOR04357作为一种潜在的药物候选物具有良好的抗体特性。
