Spiesberger Katrin, Paulfranz Florian, Egger Anton, Reiser Judith, Vogl Claus, Rudolf-Scholik Judith, Mayrhofer Corina, Grosse-Hovest Ludger, Brem Gottfried
Department of Biomedical Sciences, Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria; Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
Christian Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine Vienna, Vienna, Austria.
PLoS One. 2015 Oct 15;10(10):e0140471. doi: 10.1371/journal.pone.0140471. eCollection 2015.
30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).
With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.
Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.
30年前，人们发现双特异性抗体能够激活细胞毒性T细胞以裂解癌细胞。如今，针对多种细胞表面结构的各种双特异性抗体已被研发出来，其中大多数是在哺乳动物细胞培养系统中生产的。除了本文所述的r28M之外，尽管已经从转基因家畜的乳汁中收获了多种用于医疗需求的生物制品，但尚无已知的双特异性抗体由转基因家畜表达于血液中。在本研究中，我们调查了在转基因牛血液中表达的双特异性抗体r28M的大规模纯化及生物活性。这种串联单链可变片段抗体旨在靶向人类CD28以及黑色素瘤/胶质母细胞瘤相关的细胞表面硫酸软骨素蛋白聚糖4（CSPG4）。
采用所述优化纯化方案，从2升牛血浆中平均可获得30毫克富集的r28M组分。通过尺寸排阻色谱法将该富集组分分离为单体、二聚体和聚集体，并进一步测试其生物活性，结果表明单体组分最适合继续进行研究。对该抗体活性的详细表征证实了其诱导杀伤CSPG4阳性细胞的高特异性。此外，还初步了解了r28M激活的外周血单核细胞介导的肿瘤细胞死亡途径。考虑到其在体内的可能应用，我们还测试了向r28M中添加不同赋形剂的效果。
总之，我们成功地从牛血浆中大规模制备并纯化了单体r28M，并证明其生物活性未受影响且仍具有高度特异性，因此可能适用于黑色素瘤的治疗。
