Bessa Juliana, Boeckle Sabine, Beck Hermann, Buckel Thomas, Schlicht Sonja, Ebeling Martin, Kiialainen Anna, Koulov Atanas, Boll Björn, Weiser Thomas, Singer Thomas, Rolink Antonius G, Iglesias Antonio
Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center, Grenzacherstrasse 124, 4070, Basel, Switzerland.
Pharm Res. 2015 Jul;32(7):2344-59. doi: 10.1007/s11095-015-1627-0. Epub 2015 Jan 29.
Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations.
Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light.
The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic.
This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.
蛋白质聚集体已被视为与免疫原性相关的潜在风险因素。在此，我们开发了一种新型的人IgG转基因（tg）小鼠系统，该系统表达一小部分人IgG1抗体（Abs），用于评估人单克隆抗体制剂的免疫原性特性。
使用人Ig重链γ1（IgH-γ1）以及人Ig轻链（IgL）κ和λ基因的种系版本来生成转基因小鼠。仅使用人IgH-γ1的可溶性形式，以避免膜Ig-H链的表达以及内源性鼠Ig基因的伴随等位基因排斥。通过不同的应激条件（如与工艺相关的条件、低pH值和暴露于人工光）生成IgG1聚集体。
人Ig蛋白的表达在tg小鼠中诱导了对广泛的人IgG1分子的免疫耐受。用IgG1聚集体进行免疫表明，由大量光照诱导并携带新表位的可溶性寡聚体在tg小鼠中诱导了强烈的免疫反应。相比之下，单独的抗体聚集体和带有新表位的单体没有免疫原性。
该小鼠模型能够识别出人IgG1的免疫原性修饰。虽然不同单克隆抗体应激诱导的聚集程度有所不同，但我们使用特定单克隆抗体（单克隆抗体1）的研究结果表明，与普遍观点相反，非共价修饰的聚集体不会打破耐受性。人IgG可溶性聚集体的免疫原性潜力强烈依赖于由苛刻应激条件（即大量暴露于人工光）产生的新表位的存在。
