HOME > Research and Development Projects Adopted in FY2015 > Development of manufacturing technology on small bispecific antibody for next generation biologics
Project Leader:Asano Ryutaro
Associate Professor, Graduate School of Engineering, Tokyo University of Agriculture and Technology
We have confirmed the in vivo anti-tumor effects of Ex3 (Fig.1), a small bispecific antibody with specificity for EGFR and CD3, and also confirmed its stability enough for formulation; however, we have not yet established an efficient manufacturing process for applying it as a reagent for therapeutic use. In other words, if we develop an innovative manufacturing technology, it will be expected to accelerate a practical application of Ex3 and other small therapeutic antibodies. In this project, we will develop a manufacturing technology for small bispecific antibodies in the points of view of appropriate molecular design, optimization of microbial expression using gene sequence modification, and efficient purification using protein L. As an expression host, in addition to Escherichia coli (E. coli) and yeast which have been generally used, we will use Brevibacillus which leads to low cost production because of no endotoxin production (Fig. 2).
We have recently found that the cytotoxicity and productivity of Ex3 were improved by rearranging domain order. On the other hand, Ex3 is non-covalent dimer which has concern about dissociation during a manufacturing process and/or preservation. Although construction of single-chain diabody (scDb) format or tandem scFv (taFv) format can solve this problem, they each have eight possible domain orders and there are no reports about the comprehensive investigation of effects about domain order on function and productivity. Thus, we will try to construct expression vectors for Ex3-scDb and -taFv with different domain order, prepare using E. coli, yeast and Brevibacillus, and compare their function and expression levels. After selection of promising combinations of host cell and format, we will further try to enhance the productivity by optimization of gene sequences and establish efficient purification process using protein L. Cooperating company, ProtienExpress Co., Ltd. has own technology for functionalized protein L, which can facilitate purification of small antibodies, and is experienced in the use of Brevibacillus expression system. We will combine these technologies using Ex3 as a model seed and develop the manufacturing technology on small bispecific antibodies to accelerate their practical application.
A small bispecific antibody with specificity for EGFR and CD3 showed the marked cancer growth inhibition effects. The variations of cytotoxicity and productivity of Ex3 were found by rearranging domain order.
We first construct expression vectors for Ex3-scDb and -taFv, both structures have no concern about dissociation, and prepare them using E. coli, yeast and Brevibacillus. After selection of promising combinations of host cell and format, we will try to enhance the productivity and establish efficient purification process using protein L, and finally combine them to develop an innovative manufacturing technology on small antibodies.