Towards therapeutic antibodies to membrane oncoproteins by a robust strategy using rats immunized with transfectants expressing target molecules fused to green fluorescent protein.

Cell-surface molecules containing growth factor receptors, adhesion molecules and transporter proteins are often over-expressed in various cancer cells, and could be regarded as suitable targets for therapeutic monoclonal antibodies (mAb). Anti-cancer therapeutic mAb are claimed to bind these cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAb recognizing epitopes on the extracellular domains of native but not denatured proteins. We have experienced difficulty in obtaining mAb bound to viable cancer cells using synthetic peptides or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAb. Furthermore, we selected rats as the immunized animals, because of their excellent capacity to generate diverse antibodies. Because many target candidates are multi-pass (type IV) membrane proteins, such as 7-pass G protein-coupled receptors and 12-pass transporter proteins belonging to the solute carrier family, and their possible immunogenic extracellular regions are very small, production of specific mAb was extremely difficult. In this review, we summarize the successful preparation and characterization of rat mAb immunized against the extracellular domain of type I, type II and type IV membrane oncoproteins fused to green fluorescent protein as an approach using reverse genetics, and also introduce the discovery of cell-death-inducing antibodies as an approach using forward genetics and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen.

Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers.

Resistance of progressive cancers against chemotherapy is a serious clinical problem. In this context, human epidermal growth factor receptor 3 (HER3) can play important roles in drug resistance to HER1- and HER2- targeted therapies. Since clinical testing of anti-HER3 monoclonal antibodies (mAbs) such as patritumab could not show remarkable effect compared with existing drugs, we generated novel mAbs against anti-HER3. Novel rat mAbs reacted with HEK293 cells expressing HER3, but not with cells expressing HER1, HER2 or HER4. Specificity of mAbs was substantiated by the loss of mAb binding with knockdown by siRNA and knockout of CRISPR/Cas9-based genome-editing. Analyses of CDR sequence and germline segment have revealed that seven mAbs are classified to four groups, and the binding of patritumab was inhibited by one of seven mAbs. Seven mAbs have shown reactivity with various human epithelial cancer cells, strong internalization activity of cell-surface HER3, and inhibition of NRG1 binding, NRG1-dependent HER3 phosphorylation and cell growth. Anti-HER3 mAbs were also reactive with in vivo tumor tissues and cancer tissue-originated spheroid. Ab4 inhibited in vivo tumor growth of human colon cancer cells in nude mice. Present mAbs may be superior to existing anti-HER3 mAbs and support existing anti-cancer therapeutic mAbs.