Tornetta M, Fisher D, O'Neil K, Geng D, Schantz A, Brigham-Burke M, Lombardo D, Fink D, Knight D, Sweet R, Tsui P
Department of Molecular Discovery Technologies, Centocor Inc, Radnor, PA, USA.
J Immunol Methods. 2007 Dec 1;328(1-2):34-44. doi: 10.1016/j.jim.2007.08.008. Epub 2007 Sep 4.
The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.
治疗性蛋白质的临床开发需要能够测量蛋白质药物的药代动力学（PK）概况以及对其潜在免疫反应（IR）的检测方法。每种检测方法都需要对治疗性蛋白质具有高度特异性的试剂。对于治疗性单克隆抗体，抗互补决定区（CDR）特异性或抗独特型（抗Id）抗体是适用于这些检测的理想试剂类别，因为它们对药物抗体具有高特异性和亲和力。我们使用MorphoSys HuCAL GOLD Fab文库通过抗体噬菌体展示技术生成了针对两种人源抗体的抗Id抗体。为了选择性靶向CDR区域，在噬菌体筛选过程中加入血清和框架匹配的单克隆抗体作为竞争者。分离出了针对两个靶点的具有低至亚纳摩尔亲和力的CDR特异性Fab片段。这些Fab片段的CDR特异性通过它们与框架匹配的对照单克隆抗体不结合以及这种结合与各自治疗性单克隆抗体靶点的可溶性抗原的竞争来证明。候选抗Id Fab片段能够检测固定化和可溶性的靶标抗体，且不受血清影响，这是PK检测和IR桥接检测形式的要求。通过成对结合研究确定了用于PK检测的Fab片段组合，尽管针对一个靶标单克隆抗体的组合对PK检测缺乏所需的灵敏度。为了评估它们作为抗药物抗体（ADA）的潜力，将其中一个靶点的最佳Fab片段进行转化并制备成所需的二价人源单克隆抗体。与啮齿动物单克隆抗体和灵长类动物多克隆血清相比，噬菌体展示衍生的人源单克隆抗体与参考标准同样有效。我们的结果表明，基于竞争的噬菌体筛选可以是一种有效的方法，用于分离用于PK和IR检测开发的抗独特型抗体，并且在后一种情况下，克服了当前使用啮齿动物衍生抗Id抗体方法的局限性。
