Preliminary Study On Expression Of An Anti-CD20Antibody In CHO Cells And Site-Specific Conjugation Of MMAE To Antibody

1. Use of GC-rich element to enhance the production of anti-CD20antibody.Construction of a suitable expression vector is an effective method for improving the recombinant proteins production in CHO or other mammalian cells. Currently, there are two types of vectors most used for large-scale manufacture:DHFR-CHO system and GS-NSO system, both of which are based on technique of gene amplification to achieve the purpose of increasing the recombinant proteins production. However, the screening processes of these two systems are tedious and time consuming, and a number of reports indicate instability of recombinant protein production in DHFR-CHO system. Therefore, it is necessary to develop new high-expression vectors. Extensive literature has reported Spl-like proteins and Kruppel-like factors (KLFs) are important components of the eukaryotic cellular transcriptional machinery by regulating the expression of a large number of genes that have GC-rich promoters. Based on the above facts, we constructed a GC-rich vector by adding some GC-rich elements around the multiple cloning site of an ordinary vector. The genes of light and heavy chain equipped with a secretion signal peptide were ligated to the GC-rich vector and expressed in CHO-K1cells. The results show positive clone rate was47%(45/96) with GC-rich vector, while that of use the control vector was6%(6/96). The maximum values of OD450tested by ELISA were0.62and0.37, respectively. And the antibody titer of high expression cell clones (CHO-2F2-K2) was33mg/L, which was10times increased than our previous antibody titer. Thus, we get the following preliminary conclusions:GC-rich elements can not only increase the positive clone rate, but also improve the anti-CD20antibody expression levels in CHO cells. 2. Antibody production in suspension-cultured CHO cells.The use of animal serum in cell-culture processes brings along with it the potential for introduction of adventitious agents such as viruses and other transmissible agents. Additionally, the use of animal serum as a raw material impacts negatively on the economics of large-scale cell-culture processes. In order to further enhance the expression of the anti-CD20antibody and greatly simplify the downstream protein purification processes, the cell clone CHO-2F2-K2has been adapted to grow in serum-free suspension culture EX-CELL302, viable cell density reached1.60×106cells/ml and antibody titer reached96mg/L. Than a simple feeding strategy was employed to control the nutritional environment in the production of the antibody. The maximum viable cell density obtained was5.30x106cells/ml, a2.3-fold increase from our previous culture. And antibody titer obtained was154mg/L. Finally, the cell clone was transferred to a5-L bioreactor where the cell density reached6.40×106cells/ml and antibody titer reached200mg/L. In summary, the cell clone CHO-2F2-K2was adapted to growth in serum-free suspension culture with maximum viable cell density reached6.40×106cells/ml and antibody titer reached200mg/L, and laid the foundation for the next process optimization.3. Site-specific conjugation of MMAE to anti-CD20antibody improves the therapeutic index.Antibody-drug conjugates (ADCs), which combine the tumor-targeting capacity of monoclonal antibodies with the anti-tumor activity of cytotoxic agents, have great clinical potential for most human diseases and might play critical roles in fighting against cancer in the near future. However, traditional strategies to synthetize ADCs often yield heterogenous products with different molar ratios of drug to antibody, which brings difficulties for optimizing the biological, pharmacological, and pharmacokinetic properties of an ADC. In this study, the anti-CD20antibody2F2was engineered with cysteine substitutions at position on the heavy chains and light chains that provide reactive free-thiol group and don’t affect antibody function. The new mutant antibody is called HLM, which is expressed in CHO cells and then conjugated to monomethyl auristatin E named HLM-vcMMAE. Experiments were done to confirme the mutation and conjugation had little influence on affinity of2F2. In addition, the ADC have antiproliferative and apoptosis-inducing activity markedly superior to that of2F2and HLM.