Biochemical and Biophysical Research Communications
Structural basis for multi-specific peptide recognition by the anti-IDH1/2 monoclonal antibody, MsMab-1
Introduction
Isocitrate dehydrogenase 1 (IDH1) and IDH2 catalyze oxidative carboxylation of isocitrate into α-ketoglutarate in the cytosol and the mitochondria, respectively [1], [2]. Interestingly, several point mutations within the IDH1/2 genes have been identified in cancerous cells isolated from patients with glioma, acute myeloid leukemia, and cartilaginous tumors [3], [4], [5], [6]. Majority of these mutations occur in the same amino acid position (Arg132 in IDH1 and Arg172 in IDH2, Fig. 1A), converting them to several types of other amino acids. Especially in glioma, Arg132/Arg172 is mutated to His, Cys, Ser, Gly, Met, and Lys [7]. It has been reported that the mutated IDH1/2 shows altered enzymatic property and produces D-2-hydroxyglutarate, which increases a risk of tumor malignancy [8], [9].
Since there is a clear correlation between the mutation in the IDH1/2 and the pathogenesis, antibodies that can specifically recognize mutated but not wild-type enzymes are of great potential utility in the diagnostic or even the therapeutic applications. In fact, several monoclonal antibodies against the mutated IDH1/2 have been established and their application to the cancer diagnosis is being discussed [10].
We have isolated multiple murine antibodies that bind to the Arg-to-Gly mutant of human IDH1 (IDH1-R132G) but not to the wild type enzyme, from mice immunized with a synthetic peptide containing the mutation. One of the established antibodies, MsMab-1 (mouse IgG2a, κ), showed highly unique property in that it recognized several different IDH1 mutants without binding to the wild type version. Moreover, MsMab-1 could recognize several pathogenic IDH2 mutants without binding to the wild type IDH2 [11], [12]. The broad specificity toward multiple pathogenic IDH1/2 mutants while maintaining a strict distinction from the wild-type enzyme suggests that this antibody may be used as a single diagnostic agent to detect various IDH mutations in a cancer specimen. We have already shown that the MsMab-1 can detect mutated IDH1/2 in glioma tissues by immunohistochemical staining, indicating its high potential in diagnostic applications [10], [12]. Although the practical advantage of MsMab-1 in the cancer diagnosis is clear, it is difficult to reconcile that one antibody exhibits both highly selective and non-selective binding characters at the same time.
In this work, we performed structural analysis on the peptide binding by the MsMab-1 and clarified how the exclusive recognition of the mutated peptide was achieved while maintaining the multi-specificity.
Section snippets
IgG production and cloning
MsMab-1 was produced using mouse medial iliac lymph node methods, and its heavy and light chain genes were cloned as described previously [12]. Institutional animal committee of Tohoku University approved our study. The HA mutant of MsMab-1 was produced using a QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies Inc., Santa Clara, CA). Chinese hamster ovary (CHO)-K1 cells (American Type Culture Collection (ATCC), Manassas, VA) were transfected with the plasmids using a Gene
MsMab-1 Fab crystal structure with a mutant IDH1-derived peptide
We evaluated the binding of MsMab-1 toward various IDH1-derived 19-mer peptides by ELISA. As shown in Fig. 1B, MsMab-1 showed no binding toward wild-type peptide containing Arg132, but recognized peptides containing His, Ser, Leu and Gly at the same site (black bars). As the binding signal was the highest for the peptide with Ser substitution, we used a 12-mer peptide containing Ser mutation (designated as IDH1(Ser) hereafter) for the co-crystallization with the MsMab-1 Fab. Crystals grew under
Accession codes
Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5GIR for MsMab-1 Fab with IDH1(Ser), and 5GIS for MsMab-1 Fab with IDH2(Ser).
Funding
This work was supported in part by the “Platform for Drug Discovery, Informatics, and Structural Life Science” grant from the Japan Agency for Medical Research and Development (AMED) to J.T. and Y.Ka., by Practical Research for Innovation Cancer Control from Japan Agency for Medical Research and development, AMED (Y. Ka.), by JSPS KAKENHI Grant Number 25462242 and 16K10748 (Y.Ka.), by the Regional Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and
Acknowledgement
We would like to thank Dr. Yamashita, Dr. Higashiura, and Dr. Nakagawa at SPring-8 BL44XU for their help with X-ray data collection.
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