Evaluation of Cellular Mechanisms Involved in Recombinant Antibody Expression in Transiently Transfected Chinese Hamster Ovary Cells

Transient recombinant antibody production in Chinese hamster ovary (CHO) cells is often used to screen large panels of candidates for potential therapeutic use. However, a significant proportion of antibodies express at a level too low for adequate in vitro characterization. This study compares the pathways, including the unfolded protein response (UPR), involved in the transient expression of both low and high expressing antibody clones. Twenty historically low expressing antibodies from two different hybridoma campaigns were re-evaluated for antibody productivity using transient co-transfection of heavy and light chain plasmids in CHO 3E7 cells. Five antibodies had significantly improved antibody production while one antibody had moderately improved antibody production when expressed in CHO 3E7 cells compared to their respective historical data. Seven individual antibody chains were affected at the transcriptional level with minimal or no detectable levels of mRNA. These seven chains were used in different combinations for ten of the antibodies screened. For those ten antibodies, the lack of detectable mRNA, determined by Northern blot analysis, correlated with low levels of intracellular and secreted antibody. The low levels of mRNA could potentially be caused by enhanced degradation due to the physical characteristics of these sequences (Cooper, G. M., 2000). One variant had low levels of light chain mRNA but had secreted titers of antibody similar to its corresponding control and was the only antibody that possessed these features. The remaining four variants had adequate levels of mRNA and intracellular antibody, determined by Western blot analysis, but minimal amounts of secreted antibody, potentially implicating UPR induction. Antibodies 5L+19H (low expression) and 5L+6H (high expression) along with mock and untransfected cells were analyzed for UPR gene regulation using the Affimetrix CHO Gene array. Several observations were made. First, the transfection process alone had the most impact on differential gene expression affecting 1146 genes out of 29,700 genes assayed. Second, the UPR related gene CHOP was noted to be upregulated for both antibody transfections compared to mock, and HERPUD1 was shown to be upregulated for the low expressing antibody compared to mock. This suggests UPR induction had begun for both experimental transfections, however, the specific pathway or pathways that have been activated cannot be determined with the limited number of genes that were found to be upregulated. Additionally, when the two experimental transfections were compared to each other we found no significant difference in differential gene expression of UPR related genes. This implies that 72 hours post-transfection, the level of UPR induction was similar for an antibody that has low levels of secretion and one that has high levels of secretion. Finally, four key UPR associated genes were found to be downregulated in mock transfected cells compared to untransfected cells, (CHOP, GADD34, ERDJ4 and XBP1)

CXCR3 Depleting Antibodies Prevent and Reverse Vitiligo in Mice

Therefore, we sought to determine if targeting CXCR3 could serve as a new treatment for vitiligo