Liton Md Ferdhos Khan, Peltola Mari T, Vehniäinen Markus, Kuusela Erica, Pettersson Tiina, Lamminmäki Urpo, Pettersson Kim, Brockmann Eeva-Christine
Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland.
Department of Biochemistry/Biotechnology, University of Turku, Tykistökatu 6A, FI-20520 Turku, Finland.
J Immunol Methods. 2015 Jul;422:72-9. doi: 10.1016/j.jim.2015.04.005. Epub 2015 Apr 15.
Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome (cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys(145)-Lys(146). Due to high dissociation rate the 4D4 antibody is less than optimal for achieving a highly sensitive robust assay. In this study, we cloned the 4D4 Mab into a recombinant fragment (Fab) format and constructed three mutant libraries with the aim to increase its binding affinity. The libraries contained targeted mutations either in the CDR-H1, CDR-H2 or CDR-L3 region. PSA-I specific antibodies were enriched from the libraries by phage display technology. We identified fourteen unique clones with 1-5 mutated amino acids showing reduced dissociation of the PSA conjugate compared to the wt-4D4 Fab. Five of these mutant antibodies had 2-6 times higher binding affinity compared to the wt-4D4 Fab yet retaining the original specificity for PSA-I. The analytical sensitivity of fPSA-I assay with mutant L3-2 Fab was 0.12 μg/L compared to 4.46 μg/L with the original wt-4D4 Fab. In the method comparison study, the developed assay showed an excellent correlation to the existing fPSA-I assay. The high affinity and specificity of these mutant antibodies have potential to provide sensitive and robust detection of intact and nicked PSA from patient samples in different test formats.
前列腺特异性抗原（PSA）是前列腺癌常用的标志物。据报道，一组四种激肽释放酶免疫测定法可改善对循环中PSA升高男性前列腺活检结果（癌症与良性）的预测。其中一种激肽释放酶形式，即完整游离PSA（fPSA-I）的测定，基于一种独特的单克隆抗体（4D4），该抗体对未在Lys(145)-Lys(146)处发生内部切割的PSA具有特异性。由于解离速率高，4D4抗体对于实现高灵敏度的稳健测定并非最佳选择。在本研究中，我们将4D4单克隆抗体克隆成重组片段（Fab）形式，并构建了三个突变文库，旨在提高其结合亲和力。这些文库在互补决定区H1（CDR-H1）、互补决定区H2（CDR-H2）或互补决定区L3（CDR-L3）区域含有靶向突变。通过噬菌体展示技术从文库中富集PSA-I特异性抗体。我们鉴定出14个独特的克隆，其具有1-5个突变氨基酸，与野生型4D4 Fab相比，PSA偶联物的解离减少。其中5种突变抗体的结合亲和力比野生型4D4 Fab高2-6倍，同时保留了对PSA-I的原始特异性。与原始野生型4D4 Fab的4.46μg/L相比，突变型L3-2 Fab的fPSA-I测定的分析灵敏度为0.12μg/L。在方法比较研究中，所开发的测定法与现有的fPSA-I测定法显示出极好的相关性。这些突变抗体的高亲和力和特异性有可能在不同测试形式中对患者样本中的完整和切口PSA提供灵敏且稳健的检测。
