A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation

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Abstract

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1R132H) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1R132H, were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1R132H peptide, but not with wild type IDH1 (IDH1wt) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1R132H protein, not IDH1wt protein or the other IDH1 mutants, indicating that IMab-1 is IDH1R132H-specific. Furthermore, IMab-1 specifically stained the IDH1R132H-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1R132H-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1R132H-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.

Introduction

Gliomas are the most common primary brain tumors, and are grouped into four grades (grade I–IV) according to the World Health Organization (WHO) criteria. Among them, glioblastomas (GBMs) are the most frequent and malignant type of gliomas [1]. Despite advances in surgical techniques, radiation therapy and adjuvant chemotherapy, their prognoses remain poor: the median survival time for patients with GBMs is only one year [2]. GBMs may occur de novo (primary GBM) or may result from progression of low-grade astrocytomas (secondary GBM) [3], [4].

An unbiased, genome-wide analysis of the somatic mutations occurring in GBMs revealed recurrent mutations in arginine (amino acid position 132: R132), which is the active site of IDH1, in 12% of tumors analyzed [5]. In the subsequent genetic studies, IDH1 has been found to be mutated in 50–88% of secondary GBMs, whereas only 3–12% of primary GBMs possess these alterations [6], [7], [8], [9], [10], [11], [12], [13]. IDH1 seems to function as a tumor suppressor; therefore, mutated IDH1 contributes to tumorigenesis in part through induction of the HIF-1 pathway [14]. Analyses of progressive glioma samples, including malignant diffuse astrocytomas, anaplastic astrocytomas, well-differentiated oligodendrogliomas, anaplastic oligodendrogliomas, and the mixed oligoastrocytomas, and anaplastic oligoastrocytomas revealed that mutations in IDH1-R132 (IDH1R132H) are in fact common (>70%) in these tumors, and also occur less frequently in primary glioblastomas and other cancers [6], [7], [8], [9], [10], [11], [12], [13]. Furthermore, the IDH1 mutation was only rarely detected in any WHO grade I pilocytic astrocytomas [6], [7], [9], [15], a tumor type with infrequent malignant progression, indicating that these tumors arise through a different mechanism. A monoclonal antibody, which can specifically recognize the IDH1R132 is urgently needed for both research and clinical purposes. Furthermore, an immunohistochemical assay would be advantageous in the evaluation of the diagnosis and prognosis of a glioma biopsy as well as for biological studies such as evaluating the extent of invasion in mutation-bearing gliomas. In this study, we report a monoclonal antibody, IMab-1, which specifically detects IDH1R132H in ELISA, Western-blot analysis, and immunohistochemistry.

Section snippets

Materials and methods

Animals, cell lines, and tissues. Female BALB/c mice were obtained from The Jackson laboratory (Bar Harbor, Maine). Chinese hamster ovary (CHO) and P3U1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). CHO and P3U1 were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium including 2 mM l-glutamine (Invitrogen Corp., Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St. Louis, MO) and 1% of

Production of an anti-IDH1R132H-specific antibody

We immunized mice with synthetic peptides of IDH1-R132H mutant (IDH1R132H). After cell fusion using Sendai virus envelope, the wells of hybridomas, which were producing anti-IDH1R132H-specific antibodies, were selected in ELISA. After limiting dilution, one of the clones, IMab-1 (IgG1 subclass) was established. As shown in Fig. 1A, IMab-1 reacted with IDH1R132H peptide in a dose-dependent manner, but not with IDH1-wild type (IDH1wt) in ELISA, indicating that IMab-1 specifically recognized IDH1

Discussion

The IDH1 gene at 2q33 encodes isocitrate dehydrogenase 1 (IDH1), which catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate, which leads to the production of NADPH in the citric acid cycle [14]. IDH1 mutations occur frequently in some types of malignant gliomas, and less frequently in other gliomas [7]. To date, all mutations have been identified at the critical isocitrate binding site R132 and the vast majority of changes are heterozygous [6], [7], [8], [9], [10], [11], [12],

Acknowledgments

We thank Mr. Ling Wang, Mr. Charles Pegram, and Mr. Scott E. Szafranski for technical assistance, Dr. Joanne Ayriss, Dr. Jinli Chang, Dr. Mika Kato Kaneko, and Dr. Chifumi Kitanaka for their great help. This study was supported by NIH Grants: 5-P50-10876, 5-P50-NS-20023, CA11898, and 5-R01-CA118822. Y.K. was supported by the Global COE Program for Medical Sciences, Japan Society for the Promotion of Science.

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