Kang Seung Hoon, Jung Hahn Sun, Lee Song Jae, Park Cheon Ik, Lim Sang Min, Park Heajin, Kim Byung Sun, Na Kwang Heum, Han Gyeong Jin, Bae Jae Woo, Park Hyun Joo, Bang Keuk Chan, Park Byung Tae, Hwang Hye Seong, Jung In-Soo, Kim Jae Il, Oh Doo Byung, Kim Dong Il, Yagi Hirokazu, Kato Koichi, Kim Dae Kyong, Kim Ha Hyung
Boryung Central Research Institute, Boryung Pharmaceutical Co. Ltd., 1122-3, Shingil-dong, Danwon-gu, Ansan-si, Kyungki-do, 425-839, Republic of Korea.
Glycoconj J. 2015 May;32(3-4):161-72. doi: 10.1007/s10719-015-9590-x. Epub 2015 May 14.
Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (β1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human β1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4Ig(P)-Gal indicated that not only were the two glycans containing (β1-4)-galactose newly extended, but also glycans containing both β1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (β1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(P) is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.
人细胞毒性T淋巴细胞抗原4 - 免疫球蛋白（hCTLA4Ig）是一种免疫抑制性治疗药物，据报道，最近生产的水稻细胞来源的hCTLA4Ig（hCTLA4Ig(P)）在体外表现出与中国仓鼠卵巢细胞来源的hCTLA4Ig（hCTLA4Ig(M)）相当的免疫抑制活性。然而，hCTLA4Ig(P)的局限性包括体内半衰期缩短以及存在含有（β1-2）-木糖和α1,3-岩藻糖的非人N-聚糖，这些聚糖会在人体内引发免疫反应。在本研究中，通过人β1,4-半乳糖基转移酶（hGalT）和hCTLA4Ig的共表达来表达人β1,4-半乳糖延伸的hCTLA4Ig(P)（hCTLA4Ig(P)-Gal），试图克服这些不利影响。结果表明，编码hGalT和hCTLA4Ig均成功共表达，对纯化的hCTLA4Ig(P)-Gal中N-聚糖及其相对丰度的分析表明，不仅含有（β1-4）-半乳糖的两种聚糖被新延伸，而且与hCTLA4Ig(P)相比，同时含有β1,2-木糖和α1,3-岩藻糖的聚糖明显减少，高甘露糖型聚糖增加。与hCTLA4Ig(P)不同，hCTLA4Ig(P)-Gal作为通过（β1-4）-半乳糖进行体外唾液酸化的受体是有效的。此外，在Sprague-Dawley大鼠中静脉注射的hCTLA4Ig(P)-Gal的血清半衰期比hCTLA4Ig(P)长1.9倍，并且hCTLA4Ig(P)-Gal的清除模式与hCTLA4Ig(M)的接近。这些结果表明，hGalT与hCTLA4Ig(P)的共表达对于减少聚糖免疫原和提高体内稳定性均有用。这是关于hCTLA4Ig作为糖工程化水稻细胞中有效治疗候选物的首次报道。
