Establishment of a Multi-Specific Monoclonal Antibody MsMab-1 Recognizing Both IDH1 and IDH2 Mutations

Abstract

Mutations of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been reported in gliomas, cartilaginous tumors, and acute myeloid leukemias. IDH mutations are specific to a single codon in the conserved and functionally important arginine 132 residue (R132) of IDH1 or arginine 172 residue (R172) of IDH2 in gliomas. Although IDH1 and IDH2 catalyze the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol and mitochondria, respectively, mutated IDH1/2 proteins can possess the ability to change α-ketoglutarate to an oncometabolite R(−)-2-hydroxyglutarate. We have established several monoclonal antibodies (mAbs) specific for IDH1/2 mutations. However, no multi-specific mAb against IDH1/2 mutations has been reported. For this study, we immunized mice with an IDH1-R132G peptide of 19 amino acids (GGVKPIIIGGHAYGDQYRA), and established a novel mAb MsMab-1 that recognizes IDH1-R132G, but not wild type IDH1 in enzyme-linked immunosorbent assay (ELISA). It is particularly interesting that MsMab-1 recognizes all IDH1 mutants (R132H, R132C, R132S, R132G, R132L) in ELISA. Western blot analysis also revealed that MsMab-1 reacted with recombinant proteins of IDH1-R132H, IDH1-R132S, and IDH1-R132G, but not with wild type IDH1 and other IDH1 mutations, indicating that MsMab-1 is a multi-specific anti-mutated IDH1 mAb. Unexpectedly, MsMab-1 recognizes IDH2-R172M protein, despite that the IDH1-R132G peptide shows only 73.7% identity with the equivalent portion of IDH2-R172M (GGTKPITIGMHAHGDQYKA). Moreover, MsMab-1 stained the IDH1-R132S or IDH1-R132G-expressing glioma cells in immunohistochemistry. This report is the first to establish a multi-specific anti-mutated IDH1/2 mAb, that is expected to be useful for immunohistochemical determination of IDH1/2 mutation-bearing tumors.

A novel monoclonal antibody SMab-2 recognizes endogenous IDH2-R172S of chondrosarcoma

“…A clone RqMab-3, which was produced by immunizing synthetic peptide (NGTIQNILGG), detected both IDH1 and IDH2 (Figs. 1 and 2), and a previously established multi-specific mAb, clone MsMab-1 also detected both IDH1-R132S and IDH2-R172S [18]. An anti-IDH1-R132H mAb, clone H09 cross-reacts with IDH1-R132L [15], indicating that the cross-reaction has been often observed in anti-IDH1/2 mAbs.…”

“…Competent Escherichia coli TOP-10 cells (Life Technologies Inc.) were transformed with appropriate amounts of plasmids, pMAL-IDH1-WT, pMAL-IDH1-R132H, pMAL-IDH1-R132C, pMAL-IDH1-R132S, pMAL-IDH1-R132G, pMAL-IDH1-R132L, pMAL-IDH2-WT, pMAL-IDH2-R172K, pMAL-IDH2-R172M, pMAL-IDH2-R172W, pMAL-IDH2-R172S, and pMAL-IDH2-R140Q [18]. Then, they were cultured overnight at 37 C in LB medium (Life Technologies Inc.) containing 100 mg/ml ampicillin (Sigma-Aldrich Corp., St. Louis, MO).…”

“…To date, we established several mono-specific anti-mutated IDH2 mAbs [15]: IDH2-R172K-specific KMab-1 [16], IDH2-R172M-specific MMab-1 [16], and IDH2-R172W-specific WMab-1 [17]. We also established two multi-specific IDH mAbs: MsMab-1 [18] and MsMab-2 [19]. In this study, we report a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH.…”

A high-sensitive HMab-2 specifically detects IDH1-R132H, the most common IDH mutation in gliomas

“…In contrast, immunohistochemistry detects only one mutationbearing tumor cell; however, many types of monoclonal antibodies (mAbs) against IDH mutations should be developed. We have established two multi-specific IDH mAbs [24]: MsMab-1 [16] and MsMab-2 [19]. We also established several mono-specific antimutated IDH mAbs [24]: IDH1-R132H-specific mAbs (IMab-1 [30] and HMab-1 [18]), IDH1-R132S-specific mAb SMab-1 [31], IDH2-R172K-specific mAb KMab-1 [32], IDH2-R172M-specific mAb MMab-1 [32], and IDH2-R172W-specific mAb WMab-1 [33].…”

“…Competent Escherichia coli TOP-10 cells (Thermo Fisher Scientific Inc.) were transformed with appropriate amounts of plasmids, pMAL-IDH1-WT or pMAL-IDH1-R132H [16]. Then, they were cultured overnight at 37 C in LB medium (Thermo Fisher Scientific Inc.) containing 100 mg/ml ampicillin (SigmaeAldrich Corp.).…”