背景:哺乳动物展示是用于治疗性抗体开发的吸引人的技术。尽管哺乳动物展示的优势,例如具有哺乳动物糖基化的全长IgG展示及其选择具有良好生物物理特性的抗体的固有能力,有限的图书馆规模和大量的文化仍然是挑战。Bxb1丝氨酸整合酶通常用于将抗体基因稳定的基因组整合到哺乳动物细胞中,但是目前缺乏展示大型哺乳动物展示库所需的效率。为了提高Bxb1整合酶介导的稳定整合效率,我们的研究调查了可能影响Bxb1整合酶核定位的因素.
方法:为了提高Bxb1丝氨酸整合酶介导的整合效率,我们将各种核定位信号(NLS)融合到整合酶的N和C末端。同时,我们共表达了与核转运相关的多种蛋白质,以评估它们对编码绿色荧光蛋白(GFP)的DNA和抗体展示盒稳定整合效率的影响,这些蛋白质将整合到中国仓鼠卵巢(CHO)细胞基因组中,该细胞含有Bxb1整合酶介导的整合的着陆垫.
结果:来自非洲爪狼的核纤溶酶NLS,当与Bxb1整合酶的C端融合时,在经过测试的NLS融合中,稳定集成效率得到了最高的提高,与缺乏NLS融合的Bxb1整合酶相比,表现出超过6倍的改善。随后将额外的NLS融合物添加到Bxb1整合酶中,显示出稳定的整合效率增加了131%,其中包含了两个拷贝的C末端核质蛋白NLS融合物。通过共表达RanGTP酶激活蛋白(RanGAP)实现了进一步的改善。最后,为了验证这些发现对更复杂蛋白质的适用性,使用Bxb1整合酶与两个拷贝的C末端核纤溶酶NLS融合和RanGAP共表达,将编码膜结合临床抗体abrilumab的DNA稳定整合到CHO细胞的基因组中.与缺乏NLS融合的Bxbl整合酶相比,该方法证明整合效率增加超过14倍。
结论:本研究表明,优化Bxb1整合酶的NLS序列融合显著增强了稳定的基因组整合效率。这些发现为通过将基因稳定整合到基因组着陆垫中在哺乳动物细胞中构建更大的文库提供了实用的方法。
BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.