Improvement Of Anti-HBsAg Antibody Production In CHO Cell

Summary With the technological advances made during the past decade, antibodies now represent an important and growing class of bio-therapeutics. With the potential new targets resulting from genomics and with methods now in place to make fully human antibodies, the potential of antibodies as valuable therapeutics in oncology, inflammation and cardiovascular disease can be fully realized. Systems to produce these antibodies as full-length molecules and as fragments include expression in both mammalian and bacterial cells grown in bioreactors and in transgenic organisms. Factors including molecular fidelity and the cost of goods are critical in evaluating expression systems. Mammalian cell culture and transgenic organisms show the greatest promise for the expression of full-length, recombinant human antibodies.Generally, factors relative to antibody productivity including expression vector construction and host cell selecting and genetic reforming and cell culture in large scale and production technology. The expression vector construction is one of the most critical aspects, Including the commonly used vector making and optimization of the antibody structure and gene sequence. In the past years, the former-stuffs of this department had constructed very good eukaryotic expression vectors basing on the CHO/dhfr cell &selection-amplification system. What we did in experiment series in this report is to evaluate factors about the antibody itself which influencing the antibody expressing and production.Because the codon bias has been observed in many species. The usage of selective codons in a given gene is positively correlated with the expression efficiency. As the first experimental approach we study codon-usage effects on heterologous antibody gene expression in CHO cell, we designed and synthesized a 'hamsterized-codon-usage' human antibody gene, in which the native codons were systematically substituted with codons prevalent in highly expressed hamster genes. Relative performance of this antibody light chain gene was evaluated with CMV promoter based construct and co-expressed with high efficiency heavy chain counterpart. As a result, the modified light chain gave no significant improvement on anti-HBsAg-Ab expression, indicates that under the situation of low-level expression, the rarely-used codon does not form limiting step of antibody expression.In the second part of the study, several high-efficiency signal peptides were selected according to former reports, including hamster beta-casein (linker plus), baculovirus p67 and reconstituted light chain signal peptides to replace the original light chain signal sequence. For 4 of the light chains, after expression steps, secretion levels of antibody increased approximately 2-8-fold relative to that of the modified antibody expression construct except for the sequence with linker. The best secretion level came from the GC-content changed signal sequence. It was postulated that the translation initiation efficiency is also important to high-level expression of the antibody, because the peptide sequence is the same as the original.Due to the regulative elements in the antibody genomic sequence, it was adopted in most of presently applied antibody expression constructs. So in the 3rd part of the study, we cloned the IgGl heave chain and light chain genomic sequence, and made the CHO expression vector separately. In the following expression studies, the light chain can significantly improve the antibody secretion level, but no effects were seen in the genomic heavy chain. Our results show that the regulative sequence in the introns was involved in the antibody expression, not simply the mRNA splice mechanism. Maybe the sequence is suit for antibody promoter drived constructs in Sp2/0 cell. The effects of 5'UTR sequence was also studied with insertion of a Hsp70 5'UTR sequence, just upstream to the KOZAK sequence. We found the sequence can improve the heavy chain expression but not the light chain.A bicistronic Flp-In-CHO/ pcDNA5FRT-IRES vector was also constructed for evaluating the differences between various antibody sequences, The Flp-In System allows integration and expression of antibody gene in mammalian cells at a specific genomic location. First we introduce the ECMV-IRES sequence into the pcDNA5FRT MCS site, leaving 2 MCS sites for antibody light/heavy chain cloning. After that the anti-HBsAg-ab gene was introduced, the expression of different stable clone produce was assayed with ELISA. The variation of the antibody secretion level of these clone was in 40%. Sequences relative to hEFIA locus control region were cloned for the the regulation analysis.In conclusion, the experiments studied factors influencing antibody expression level around the antibody sequence systematically, that will form a strong basis for high-level antibody expression vector construction.