(228cb) Rare Residues Mutation Approach for Changing Specificity of Antibody

Glioma is a type of tumor formed in brain, and mutations at a functional arginine residue of isocitrate dehydrogenase 1/2 (IDH1/2) in glioma have been reported. In the case of IDH1, mutations of the arginine 132 residue (R132) of IDH1 in glioma have shown good prognosis of patients ^[1]. Therefore, the R132-mutated IDH1s can be a promising prognostic marker in glioma. Antibodies specific for the R132-mutated IDH1 are powerful tools for glioma diagnosis, however developing antibodies to all of the IDH1 mutants is challenging. Recently, multi-specific anti-mutated IDH1 monoclonal antibody, termed MsMab-1, has been generated ^[2]. This antibody showed specific binding to four R132-mutated IDH1 (R132H, R132S, R132G and R132L), enabling detection of multiple IDH1 mutations by a single antibody. In this study, we aim to develop an antibody that can detect all of the R132-mutated IDH1s by improving multi-specificity of MsMab-1 by directed evolution.

We first constructed a single chain fragment of variable region (scFv) format of MsMab-1 antibody that allows us to perform high throughput functional analysis of MsMab-1 mutants. We transformed E. coli with the vectors encoding scFv of MsMab-1. The specificity of MsMab-1 scFv against various R132-mutated IDH1 was measured by sandwich ELISA. As a results, MsMab-1 scFv showed specificity against four R132-mutated IDH1, indicating that MsMab-1 scFv retained the multi-specificity of its parental antibody, MsMab-1 IgG.

Next, we attempted to improve multi-septicity of MsMab-1 scFv by a site-specific mutagenesis. Because MsMab-1 antibody has an unique molecular recognition mechanism, we focused on unusual amino acid residues of MsMab-1 compared to other antibodies. We prepared all mutants at these amino acid residues, respectively. High throughput functional analysis revealed that Cys 47 residue in VH was critical for multi-specificity of MsMab-1. Unexpectedly, a C47N mutant showed improved affinity and multi-specificity. These results suggest that rare residue in variable domains influences specificity of antibody.

Together, we succeeded in preparing MsMab-1 scFv and found that rare residues influenced the specificity of MsMab-1. We are currently working on further improving multi-specificity by introducing mutations at multiple rare residues.

[1] H.Yan et al., New England Journal of Medicine, 360, 763-73, 2009.

[2] Y. Kato, Brain Tumor Pathology, 32, 3-11, 2015.