Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Structural basis for multi-speciﬁc peptide recognition by theanti-IDH1/2 monoclonal antibody, MsMab-1
Yu Kitago a, Mika K. Kaneko b, Satoshi Ogasawara b, Yukinari Kato b, **, Junichi Takagi a, *ab

Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 10 August 2016
Accepted 18 August 2016
Available online 20 August 2016

A point mutation in isocitrate dehydrogenase 1 (IDH1) and IDH2 is directly linked to the pathogenesis ofcertain types of tumors. To detect this mutation, several antibodies that can distinguish between mutant
and wild-type enzymes have been established. One of which, MsMab-1, has a unique multi-speciﬁccharacter against several types of mutated IDH1/2. This promiscuous character is in remarkable
contrast to the highly speciﬁc antigen recognition typically observed with a monoclonal antibody. We
solved the crystal structure of MsMab-1 Fab fragment in complex with either IDH1 or IDH2-derived
peptides. Based on the structure, it became clear that the peptide-binding pocket of the antibody ishighly complementary to the core determinant shared between the IDH1 and IDH2, while leaving justenough space for the side chain of the pathogenic but not the wild-type amino acids located in themutation position. Clariﬁcation of the molecular basis for the peculiar binding characteristics of MsMab1 in atomic detail will help facilitating its diagnostic application, and may be used to develop better
diagnostic reagents through structure-guided protein engineering.
© 2016 Elsevier Inc. All rights reserved.

Keywords:
Monoclonal antibody
X-ray crystallography
Multi-speciﬁcity
Isocitrate dehydrogenase
Tumor diagnosis

1. Introduction
Isocitrate dehydrogenase 1 (IDH1) and IDH2 catalyze oxidative
carboxylation of isocitrate into a-ketoglutarate in the cytosol andthe mitochondria, respectively [1,2]. Interestingly, several point
mutations within the IDH1/2 genes have been identiﬁed incancerous cells isolated from patients with glioma, acute myeloid
leukemia, and cartilaginous tumors [3e6]. Majority of these mutations occur in the same amino acid position (Arg132 in IDH1 andArg172 in IDH2, Fig. 1A), converting them to several types of otheramino 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-2hydroxyglutarate, 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 speciﬁcallyrecognize mutated but not wild-type enzymes are of great potential
utility in the diagnostic or even the therapeutic applications. In fact,* Corresponding author.
** Corresponding author.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp (Y. Kato), takagi@protein.osaka-u.
ac.jp (J. Takagi).
http://dx.doi.org/10.1016/j.bbrc.2016.08.110
0006-291X/© 2016 Elsevier Inc. All rights reserved.

several monoclonal antibodies against the mutated IDH1/2 havebeen established and their application to the cancer diagnosis isbeing discussed [10].
We have isolated multiple murine antibodies that bind to the
Arg-to-Gly mutant of human IDH1 (IDH1-R132G) but not to thewild type enzyme, from mice immunized with a synthetic peptide
containing the mutation. One of the established antibodies,
MsMab-1 (mouse IgG2a, k), showed highly unique property in thatit recognized several different IDH1 mutants without binding to thewild 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Y. Kitago et al. / Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

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2.3. Fab preparation and crystallization

Fig. 1. Promiscuous recognition of IDH-derived peptide by MsMab-1. A, Amino acidsequences of human IDH1 and IDH2 near the pathogenic mutation position. The regions that are directly in contact with MsMab-1 is shaded in gray, with the two residues that differ between IDH1 and IDH2 shown in red. B. The binding ability of
MsMab-1 against various peptides conﬁrmed by ELISA. Binding of wild-type (ﬁlledbars) or HA mutant (open bars) versions of MsMab-1 IgG toward wells coated with 19mer IDH1-derived peptides with indicated mutation was evaluated. Uncoated wells areused as control. Data are mean ± SD (n ¼ 3) from a representative experiment. (Forinterpretation of the references to colour in this ﬁgure legend, the reader is referred to
the web version of this article.)

multi-speciﬁcity.
2. Materials and methods

To produce Fab fragment, MsMab-1 IgG was digested by papain(Papain-agarose: Thermo Fisher Scientiﬁc Inc.) in PE buffer (20 mM
potassium sodium phosphate buffer pH 7.0, 10 mM EDTA) containing 25 mM Cys-HCl pH 7.0. After dialysis against TBS buffer(20 mM Tris-HCl pH 8.0, 150 mM NaCl), Fc fragment was removed
from the digested mixture by Protein A-Sepharose (GE Healthcare),and the ﬂow through fraction was desalted on a PD-10 column (GEHealthcare) and concentrated to ~10 mg/ml with an Amicon Ultra15 ultraﬁltration membrane (30 kDa MWCO; EMD Millipore Corp.,
Billerica, MA) before crystallization. For crystallization of thecomplex with a ligand peptide, 1/10 volume of 10 mM peptide
dissolved in water was added to the puriﬁed Fab sample just before
the crystallization. Synthetic peptides IDH1(Ser) (KPIIIGSHAYGD)
and IDH2(Ser) (TKPITIGSHAHGDQYK) were purchased from SigmaAldrich Corp.
The crystallization screening of the IDH1(Ser)-Fab complex was
performed using the sitting drop vapor diffusion method with theIndex (Hampton Research, Aliso Viejo, CA) and Classics Neo (Qiagen, Hilden, Germany) kits. The initial condition was optimized,
and diffraction-quality crystals were obtained under the conditionof 0.1 M imidazole buffer pH 6.5 containing 2.0 M ammonium
sulfate, 0.2 M potassium sodium tartrate with the hanging drop
vapor diffusion method. The crystal for the IDH2(Ser)-Fab complex
was obtained under the same condition. For the diffraction datacollection, the crystals were soaked in the same solution containing
10%(v/v) ethylene glycol, and was rapidly frozen with liquid
nitrogen.
2.4. X-ray data collection and processing

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 Pulser Xcell electroporation system (Bio-Rad Laboratories
Inc., Berkeley, CA), and recombinant HA mutant of MsMab-1 was
puriﬁed using Protein G-sepharose (GE Healthcare, Chicago, IL)from culture supernatant.

The diffraction data sets were collected at BL44XU beamline,
SPring-8 (Hyogo, Japan), and the diffraction images were processedwith XDS [13]. For the Fab-IDH1(Ser) complex, the initial phases
were calculated by molecular replacement method with PHASER[14] using a Fab structure of mouse IgG2a (PDID: 4EBQ) as a searchmodel. The initial phases were then improved by density modiﬁcation with DM [15], and an initial model was automatically builtwith ARP/wARP [16]. The model was reﬁned with phenix.reﬁne [17]and iterative manual editing using COOT [18]. For IDH2(Ser)-Fabcomplex, the initial phases were calculated by molecular replacement method using the structure of IDH1(Ser)-Fab as a searchmodel, and the resultant model was reﬁned with manual editing on
COOT and REFMAC5 [19]. The model validation was carried out with
MOLPROBITY [20], and all ﬁgures were prepared with PyMOL(http://www.pymol.org/). Diffraction data statistics and ﬁnal
reﬁnement statistics are shown in Supplement Table 1.

2.2. Enzyme-linked immunosorbent assay (ELISA)

3. Results and discussion

Synthetic peptides (Sigma-Aldrich Corp., St. Louis, MO) were
immobilized on Nunc Maxisorp 96-well immunoplates (Thermo
Fisher Scientiﬁc Inc., Waltham, MA) at 1 mg/ml for 30 min. Synthetic
peptides are as follows: GGVKPIIIGRHAYGDQYRA (IDH1-WT),GGVKPIIIGHHAYGDQYRA (IDH1-R132H), GGVKPIIIGSHAYGDQYRA(IDH1-R132S),GGVKPIIIGGHAYGDQYRA(IDH1-R132G),GGVKPIIIGLHAYGDQYRA (IDH1-R132L). After blocking with SuperBlock T20 (PBS) Blocking Buffer in PBS (Thermo Fisher ScientiﬁcInc.), the plates were incubated with puriﬁed mAbs (1 mg/ml), followed by an incubation with peroxidase-conjugated anti-mouse
IgG (1:1000 dilution, Agilent Technologies, Inc.). The enzymatic
reaction was conducted with 1-Step Ultra TMB-ELISA (Thermo
Fisher Scientiﬁc Inc.). The optical density was measured at 655 nm
using an iMark microplate reader (Bio-Rad Laboratories Inc.).

3.1. MsMab-1 Fab crystal structure with a mutant IDH1-derived
peptide2.1. IgG production and cloning

We evaluated the binding of MsMab-1 toward various IDH1derived 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 thesame 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 cocrystallization with the MsMab-1 Fab. Crystals grew under multiple conditions, and we successfully obtained diffraction quality
crystals after optimization. From the diffraction dataset collectedusing the BL44XU at SPring-8, initial phases were calculated by

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Y. Kitago et al. / Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

molecular replacement method, and the ﬁnal structure was reﬁned
at the resolution of 1.93 Å. The crystal contained two copies of
peptide-Fab complexes in the asymmetric unit. Electron densities
were clearly visible for all 12 residues of IDH1(Ser) peptide boundto one Fab (molecule 1) in the asymmetric unit, while the C-terminal 2 residues of the peptide bound to the other Fab (molecule 2)
were disordered. Nevertheless, the visible 10 residues had the same
conformation in the two molecules. As the variable region of theantibody molecules were also virtually identical, we will only
discuss the structure of the molecule 1 hereafter. We will also use
the peptide residue numbering based on their original IDH
sequence positions throughout the paper.
The MsMab-1 Fab fragment assumes a typical Fab structure
consisting of four immunoglobulin-like b-sandwich domains, andthe ligand peptide is packed into a pocket formed by CDR loops L2,
L3, H1, and H3 (Fig. 2A). The peptide sits on a bent H3 (Fig. 2B), whichis exceptionally short (4 residues) compared to typical H3 loops inthe database [21,22]. This feature made the antigen binding groove
narrow and deep, burying most of the surface of the antigen peptide.
The N-terminal half of the peptide has extended conformation andlies along the narrow cleft between heavy and light chains. At Gly131
in the middle, the peptide backbone bends abruptly and forms single
turn helix ascending from the bottom of the cleft (Fig. 2B), andprojects the C-terminal two residues that are disordered in themolecule 2 out of the pocket (Fig. 2C, asterisk). The side chains of
peptide residues Ile128, Ile130, His133, and Tyr135 all made intimate
VDW contacts with the surface of Fab (Fig. 2C, 2D), indicating theirfundamental contribution to the interaction. The presence of Gly at
the position 131 also seems essential, since its Ca atom is in a directcontact with the ﬂoor of the cleft and presence of any side chain
would not be tolerated (Fig. 2D). The side chain of the pathogenic
mutation residue, Ser132, is pointing toward a cavity formed by the
Fab and the Ile129 of the peptide itself, with its hydroxyl oxygen
making a direct hydrogen bond with His95 (based on the Chothia's
numbering scheme, [23]) of antibody heavy chain (Fig. 2C).
In order to simulate the binding of various IDH1 peptides, we
modeled different side chains for the residue 132 based on the
crystal structure of IDH1(Ser)-Fab complex (Fig. 3). When themutations shown to be permissive for the MsMab-1 binding wereintroduced, the side chains were well accommodated in the cavity.
However, the wild-type residue Arg is too large and expected tocause steric clash with either the antibody or the peptide itself.
Therefore, the multi-speciﬁc property of MsMab-1 as well as its
inability to recognize wild-type IDH1 is warranted by creating aright size of cavity through a unique cooperation between antigen
binding pocket and the bound peptide itself.

Fig. 2. Structure of MsMab-1 in complex with IDH1(Ser) peptide. A, IDH1(Ser) peptide (stick model with CPK coloring) recognized by MsMAb-1 Fab (surface rendering) is shown.
L2, L3, H1, and H3 loops are color-coded as indicated. H2 and L1 loops do not make contact with the peptide. B. An alternative view of A. Fab and IDH1(Ser) are shown as ribbon anda thick tube, respectively, with the same color-code as in A. The backbone of IDH1(Ser) bends abruptly at the Gly131 (red sphere). C, D. The close up views of the binding site near the
Ser132. In C, a hydrogen bonding interaction between Ser132 and His95H is shown as a green dashed line. The last two residues of IDH1(Ser) Gly136 and Asp137 (asterisk) aredisordered in one of the complex in an asymmetric unit. In D, only the peptide segment corresponding to residues 127e135 is shown. The heavy chain and light chain surface of Fab
are colored by light blue and pink, respectively. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)Y. Kitago et al. / Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

1277

Fig. 3. Docking simulation of various IDH1 mutant peptides. In order to simulate the binding of various mutant IDH1 peptides, crystal structure of MsMab-1 Fab is shown in asurface model with CPK color scheme with the bound peptide portion modiﬁed to remove the Ser132 side chain (i.e., equivalent to Ser to Gly mutation) rendered in yellow surface.
The top panel represents the actual crystal structure obtained with IDH1(Ser) where Ser side chain is shown in stick model. The bottom four panels each represent hypothetical
docking model of peptide containing various amino acid residues including Gly, His, Leu, and Arg (¼ wild type) at position 132. (For interpretation of the references to colour in thisﬁgure legend, the reader is referred to the web version of this article.)

3.2. Ala substitution for His95 of heavy chain changes ligand
recognition property

3.3. Crystal structure of MsMab-1 Fab in complex with mutated
IDH2-derived peptide

On the Fab side, the H3 loop residue His95 constitute the wall of
the cavity described above, and deﬁnes its volume (Fig. 3). We
speculated that changing the side chain of His95 to a smaller one
may enlarge the cavity, enabling a design of mutant antibody with abroader speciﬁcity. To test this hypothesis, we prepared a recombinant mutant MsMab-1 containing Ala in place of His95 (HAmutant). The HA mutant version of MsMab-1 IgG was stably
expressed using CHO-K1 cells, and puriﬁed from the culture supernatant (with a typical yield of 0.5 mg from 2 L media). In a
separate experiment, we also conﬁrmed that the original wild-type
MsMab-1 can be recombinantly produced at similar level (data now
shown), indicating that the mutation does not affect the stability of
the antibody. The puriﬁed HA MsMab-1 was evaluated for thebinding toward different IDH1 peptides by using the same ELISA
format as the original MsMab-1 (Fig. 1B, white bars). Contrary to
our expectation, the mutant antibody did not show broader speciﬁcity than the original antibody; rather, the speciﬁcity became
narrower because the HA MsMab-1 could only bind peptides carrying Gly and Ser at the position 132. We note that Tyr32 in the H1loop sits right next to the His95, with its side chain phenolic ringpushed back by the His95 imidazole (Fig. 3). Retrospectively, thevacancy created by the removal of the His95 side chain would bereadily ﬁlled by that of Tyr32, making the cavity even smaller.
Although our simple subtraction strategy of structure-guided reengineering to obtain an antibody with a desired property did notwork, the result conﬁrmed the essential role of His95 in discriminating amino acid types present in the residue 132 of IDH1, validating our crystal structure.

The remaining puzzle about the promiscuous binding propertiesof MsMab-1 is its ability to recognize pathogenic IDH2 peptide
whose sequence is similar but distinct from that of IDH1. Within the
10-residue region of IDH1 peptide making direct contact with the
MsMab-1, sequence variations exist at two positions; the Ile129 andTyr135 in IDH1 are replaced by Thr169 and His175 in IDH2, respectively (Fig. 1A). The ﬁrst position (Ile129) exposes its side chaingroup to the solvent, suggesting that the Thr residue of IDH2 isallowed in this position (Fig. 2D). However, the side chain of thesecond position (Tyr135) contributes signiﬁcantly to the interaction,
by a hydrophobic packing with the side chain of Ile55 from the L2loop as well as a water-mediated hydrogen bonding network
involving main chain carbonyls (Fig. 4A). To understand themechanism underlying the dual-speciﬁcity, we crystalized MsMab1 Fab in the presence of a 16-mer IDH2-derived peptide containing
a pathogenic Arg-to-Ser mutation at the residue 172 (i.e.,
IDH2(Ser)). From the crystal structure solved at a resolution of
1.93 Å, it became clear that the antibody indeed recognized IDH2derived peptide in a manner indistinguishable from that of IDH1
peptide (Fig. 4B). First, the Fv part of the antibody superimposeswith that of the IDH1(Ser)-complex with an RMSD value of 0.39 Å
for a total of 215 residues, and virtually no difference is observed forthe conformation of the side chains of all residues making directcontacts with the ligand peptides (Fig. 4C). This indicates that theantibody does not adjust its structure for the recognition of two
different peptides. At the peptide side, the central 10 residues(Pro167-Gly176) are visible in the electron density map. When superposed onto the Fab-IDH1(Ser) complex structure using the Fv

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Y. Kitago et al. / Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

Fig. 4. Structural comparison between MsMab-1 bound by IDH1(Ser) and IDH2(Ser). A, B, The close-up views of the recognition environment for the isotype-speciﬁc residue
Tyr135 in IDH1 (A) and His175 in IDH2 (B). Water molecules (blue spheres) and hydrogen bonds (pink dashed lines) are shown. A and B are viewed from the same orientation after
structurally aligning the Fv portions. C, Superposition of the two complex structures. The antibody residues making direct contacts with IDH1(Ser) peptide (yellow tube) are shownin blue (H chain) and red (light chain) stick models, respectively, and the corresponding residues in the IDH2(Ser) (green tube) complex are in white color. D, Conformation of theIDH1(Ser) (yellow) and IDH2(Ser) (green) peptides bound onto MsMab-1 (Ca trace). Only the peptide segments that are present in both structures are shown. Residues that differ
between the isoforms (Tyr135/His175 and Ile129/Thr169) are indicated by red dashed circles. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to
the web version of this article.)

portion, the two peptides have essentially identical conformation,
showing an RMSD value of 0.20 Å for 10 matched Ca atoms. As
expected, Thr169 is pointing away from the antibody, consistent
with the lack of peptide discrimination at this position (Fig. 4D). For
the second subtype-speciﬁc residue, the His175 perfectly emulates
conformation of the Tyr135 and keep the same ring stacking with
the Ile55L (Fig. 4A,B,D). Furthermore, acquisition of an additional
water restored the hydrogen bonding network, making the interface virtually identical despite the Tyr to His replacement (Fig. 4B).
Thus, we conclude that MsMab-1 accomplishes the dual speciﬁcity
by recognizing common structural feature shared between IDH1
and IDH2. Furthermore, the structural environment of the pathogenic mutation residue is conserved between IDH1 and IDH2,
explaining why MsMab-1 exhibits promiscuity (i.e., the dual
recognition and the large choice of amino acid at the mutation
position) without recognizing the wild-type IDH1/2 proteins.
The point mutation at Arg132 of IDH1 and Arg172 of IDH2 iscommonly observed in 50e80% of gliomas [7]. A rapid detection ofthis mutation in a patient is necessary to enable accurate diagnosis
required for designing appropriate therapeutic strategies. Particularly, the diagnosis by the immunohistochemical staining of a pathological tissue sample is considered superior to the genetic analysis of
the patient, because it can be implemented more easily in a wide
variety of clinical situations. Although the potential of using theimmunohistochemistry in the diagnosis of gliomas has been

postulated [24], there is one drawback to this method. So far, speciﬁc
antibodies for several pathogenic mutated IDHs have been developed
and commercially available. However, since over ten types of pathogenic mutation at the key Arg residue have been reported on both
IDH1 and IDH2, one must search for many different antigens in atissue specimen. So far, antibodies speciﬁc for several pathogenic
mutated, but not wild-type, sequences of IDH have been developed
and commercially available. Each antibody was raised against individual mutant sequence, and must be used in combination or as a
mixture to detect unknown mutation. In contrast, the MsMab-1 canbe used alone to detect many disease-causing mutations in both IDH1
and IDH2, simplifying the diagnostic procedure. Although this property was counter-intuitive due to the well-established antibody's role
as a highly selective binder, our structural analyses unraveled themechanism underlying this seemingly puzzling phenomenon. It is
anticipated that the structural information obtained in this study canbe used to engineer better antibodies with improved afﬁnity and/or
modiﬁed speciﬁcity, contributing to the development of easy andaccurate diagnostic tools for human glioma.
Accession codes
Coordinates and structure factors have been deposited in theProtein Data Bank under accession codes 5GIR for MsMab-1 Fabwith IDH1(Ser), and 5GIS for MsMab-1 Fab with IDH2(Ser).

Y. Kitago et al. / Biochemical and Biophysical Research Communications 478 (2016) 1274e1279

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 Technology (MEXT) of Japan(Y.Ka.), and by the Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED (Y.Ka.). This work was
performed under the Cooperative Research Program of Institute forProtein Research, Osaka University (CR15-05).
Acknowledgement
We would like to thank Dr. Yamashita, Dr. Higashiura, and Dr.
Nakagawa at SPring-8 BL44XU for their help with X-ray datacollection.
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2016.08.110.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2016.08.110
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