CHO-GMT recombinant protein expression
A technology of COS-7 and glycoprotein, applied in the field of biotechnology and molecular biology, can solve problems such as difficult mass production and expensive chemical synthesis
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Embodiment 1
[0162] CHO glycosylation mutants can be used to generate recombinant MUC1 with tumor-associated O-glycans
[0163] Chinese hamster ovary (CHO) cells have become a major host cell for the production of recombinant glycoproteins in the biotechnology industry. N- and O-glycosylation in CHO cells has been a major research focus for many years. O-glycosylation pathways can be complex and cell-type specific in different mammalian cells (Tarp and Clausen, 2008). However, O-glycosylation in CHO cells is straightforward and has a core 2 to core 1 shift. O-glycans on recombinant erythropoietin (EPO) produced by CHO cells exist in only two forms: ST and diST (Hokke et al., 1995). Recombinant MUCl produced by CHO cells also mainly contains ST and diST (Backstrom et al., 2003; Olson et al., 2005; Rughetti et al., 2005). Detailed analysis showed that O-glycans produced by CHO cells linked to recombinant MUCl contained 85% ST, 13% diST and only 2% T (Rughetti et al., 2005). All O-glycans...
Embodiment 2
[0183] MUC1 glycopeptides with tumor-associated O-glycans trigger cancer-specific MUC1 antibody responses. Mannose-terminated N-glycans on MUC1 enhance its potency by enhancing DC and macrophage uptake.
[0184] To study the efficacy of the recombinant N-terminal subunit of MUC1 as a therapeutic vaccine in mouse models and breast cancer patients, the following experimental procedure was used:
[0185] 1. Generation of MUCl-Fc fusion protein.
[0186] a. Generation of expression constructs to express the MUCl-Fc fusion protein. MUCl (N-terminal subunit) and Fc are linked by a cleavable linker.
[0187] b. Production of MUCl-Fc in CHO-gmt5 and CHO-gmt6 cells. For human pilot studies, this production will be done by the inventor's industrial collaborators with GMP facilities.
[0188] 2. Mouse studies
[0189] a. Tumor dynamics study. Establishment of MUC1 mammary tumors in MUC1 transgenic mice. These mice were immunized with liposome formulations of the N-terminal subunit...
Embodiment 3
[0259] GDP-fucose transporter mutant
[0260] It is widely believed that removal of fucose from the N-glycan linked to Asn297 of human IgG 1 significantly enhanced its binding to its receptor FcYllla, and thus markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) (12;13) . Recent reports have shown that removal of sialic acid from IgG1 also enhances ADCC in vivo (14; 15). As disclosed above, the present inventors generated a CHO mutant line (16) with a dysfunctional CMP-sialic acid transporter. Thus, the glycoproteins produced by this cell line do not contain sialic acid at all. The mutant line was initially named MAR-11, which was later renamed CHO-gmt1, CHO glycosylation mutant-1. The present inventors used zinc finger nuclease technology and successfully knocked out the gene encoding GFT from CHO-gmt1 cells. The resulting cell line, CHOgmt5, has been shown to produce N-glycans substantially sparing sialic acid and fucose. The ultimate aim of the inventors w...
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