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Humanization and Epitope Mapping of
Neutralizing Anti-Human Fas Ligand
Monoclonal Antibodies: Structural Insights
into Fas/Fas Ligand Interaction
Tsukasa Nisihara, Yoshitaka Ushio, Hirohumi Higuchi,
Nobuhiko Kayagaki, Noriko Yamaguchi, Kenji Soejima,
Seishi Matsuo, Hiroaki Maeda, Yasuyuki Eda, Ko Okumura
and Hideo Yagita
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:3266-3275; ;
doi: 10.4049/jimmunol.167.6.3266
http://www.jimmunol.org/content/167/6/3266
Humanization and Epitope Mapping of Neutralizing
Anti-Human Fas Ligand Monoclonal Antibodies: Structural
Insights into Fas/Fas Ligand Interaction1
Tsukasa Nisihara,* Yoshitaka Ushio,* Hirohumi Higuchi,* Nobuhiko Kayagaki,†‡
Noriko Yamaguchi,†‡ Kenji Soejima,* Seishi Matsuo,* Hiroaki Maeda,* Yasuyuki Eda,*
Ko Okumura,†‡ and Hideo Yagita2†‡
F
as ligand (L)3/CD95L is an integral type II membrane protein belonging to the TNF family (1, 2). Expression of
FasL is observed in a variety of tissues and cells such as
activated lymphocytes and the anterior chamber in the eye. FasL
induces apoptotic cell death in various types of cells via its corresponding receptor, Fas/CD95/APO1. FasL not only plays important roles in immune surveillance against transformed cells and
homeostasis of activated lymphocytes, but it has also been implicated in establishing immune-privileged status in the testis and eye
(1– 4). Despite these facts supporting physiological importance of
FasL-induced apoptosis, recent studies have indicated that the
FasL/Fas system is also involved in the pathogenesis of various
diseases such as fulminant hepatitis, graft-vs-host disease
(GVHD), and AIDS (1, 5–7).
To date, at least 18 molecules, including TNF-␣, lymohotoxin
(LT)-␣/TNF-␤, FasL, LT-␤, CD27L, CD30L, CD40L, 4-1BBL,
OX40L, TNF-related apoptosis-inducing ligand (TRAIL)/APO2L,
TNF-like weak inducer of apoptosis/APO3L, and B cell activation
factor from the TNF family/NH2-terminal kinase/B lymphocyte
stimulator, have been reported to be the TNF family members (8,
9). All these members, except for LT-␤, exist as type II cell surface
proteins and exhibit pleiotropic biological activities such as cell
*The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan; †Department of
Immunology, Juntendo University School of Medicine, Tokyo, Japan; and ‡Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan
Received for publication May 16, 2001. Accepted for publication July 5, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Science and Technology Agency, the
Ministry of Education, Science, and Culture, and the Ministry of Health, Japan.
2
Address correspondence and reprint requests to Dr. Hideo Yagita, Department of
Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper used in this paper: L, ligand; GVHD, graft-vs-host
disease; LT, lymphotoxin; TRAIL, TNF-related apoptosis-inducing ligand; CRD, cysteine-rich domain; CDR, complementarity-determining region; FR, framework region; DSS, disuccnimidyl suberate; hFas, human Fas; CHO, Chinese hamster ovary.
Copyright © 2001 by The American Association of Immunologists
death, differentiation, and proliferation via their binding to cognate
receptors belonging to the TNFR family that have several repeats
of characteristic cysteine-rich domain (CRD) in their extracellular
region (8 –10). Like other members of the TNF family of molecules, the biologically active FasL molecule exists as a homotrimerized complex (11). A FasL trimer recruits three Fas molecules,
resulting in oligomerization of a cytoplasmic death domain, which
recruits and activates procaspase-8 via an adaptor molecule, Fasassociated death domain. The activated caspase-8 in turn activates
downstream caspases that lead to apoptotic cell death characterized by structural changes such as DNA fragmentation and chromatin condensation (1).
Among the TNF family members, the crystal structures of
TNF-␣, LT-␣, CD40L, and TRAIL have been revealed (12–18).
Despite relatively low amino acid sequence similarity between
these molecules, their three-dimensional structures are highly conserved. Each monomer consists of sandwich (jellyroll) topology
composed of several ␤ strands and loops bridging the ␤ strands.
Further studies of the crystal structures of LT-␣/TNFRI and
TRAIL/TRAIL-R2 complexes revealed that one individual receptor molecule interacts with two ligand molecules in the trimer
complex via two different contact sites, one between the outer
tip-forming D-E loop on the ligands and the loop motif on the
second CRD in the receptors and another between the residues
relatively near the top of the ligands and the loop on the second/
third CRD of the receptors (18, 19).
Previously, we established murine neutralizing anti-human FasL
mAbs, NOK1, NOK2, and NOK3, and characterized the biological
nature of FasL (20). Furthermore, we indicated the therapeutic
usefulness of neutralizing anti-FasL mAb in murine models of fulminant hepatitis and lethal acute GVHD (6, 7). In the present
study, we generated a humanized version of NOK2, which might
be useful for clinical application to human diseases. By extensive
alanine-scanning mutagenesis and computed molecular modeling,
we determined amino acid residues on human FasL that constitute
the conformational epitopes recognized by murine NOK1, -2, and
-3 and humanized NOK2. We also determined which human FasL
residues are critical for Fas binding and cytotoxic activity. Based
0022-1767/01/$02.00
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Fas ligand (L)/CD95L, a proapoptotic member of the TNF family, is a potential target for clinical intervention in various diseases.
In the present study, we generated a humanized anti-human FasL mAb and characterized the epitopes of neutralizing mAbs by
extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs
recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E
loop and another near the top of FasL. Both of these sites on FasL are critically involved in the direct interaction with the
corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these
FasL/Fas contact sites. The Journal of Immunology, 2001, 167: 3266 –3275.
The Journal of Immunology
on these results, a molecular model of FasL/Fas interaction is
proposed.
Materials and Methods
Cells
Chinese hamster ovary (CHO) cells and COS cells were obtained from
American Type Culture Collection (Manassas, VA) and cultured in RPMI
1640 medium containing 10% FCS, 100 ␮g/ml streptomycin and penicillin, and 2 mM glutamine (culture medium). WR19L cells and human Fas
(hFas) cDNA transfectants (hFas/WR19L) were kindly provided by Dr. S.
Yonehara (Kyoto University, Kyoto, Japan) and cultured in the culture
medium.
Reagents
Murine anti-human FasL mAbs, NOK1 (IgG1-␬), NOK2 (IgG2a-␬), and
NOK3 (IgM-␬), were purified from culture supernatants as previously described (20). An anti-FLAG mAb M2 and disuccnimidyl suberate (DSS)
were purchased from Eastman Kodak (New Haven, CT) and Pierce (Rockford, IL), respectively.
Humanization of NOK2
Construction and expression of FasL mutants
The cDNA encoding the extracellular domain of FasL was amplified by
RT-PCR from pMKITNeo (kindly provided by Dr. K. Maruyama, Tokyo
Medical and Dental University, Tokyo, Japan) containing full-length human FasL (20) by using TCT GGT ACC TGT GGG CAG CTC GAC TAC
AAG GAC GAC GAT GAC AAG CAC CTA CAG AAG GAG CTA GCA
GAA CTC CGA GAG TCT as the 5⬘ primer and GCC AAG CTT GGA
TCC TTA GAG CTT ATA TAA GCC GAA as the 3⬘ primer. The KpnI
site plus the FLAG tag sequence and the BamHI site were added to the 5⬘
primer and the 3⬘ primer, respectively. After digestion with KpnI and
BamHI, the PCR product of 577 bp was subcloned into the KpnI and
BamHI sites of pCAGn-C25VL, which has been constructed to express the
V␬ region of anti-HIV mAb C25 under the control of chicken ␤-actin
promoter, rabbit ␤-globin splicing acceptor, and human CMV enhancer
(details will be described elsewhere), resulting in in-frame fusion of the
human Ig signal sequence, the FLAG tag, and the extracellular domain of
human FasL (native hFasL/pCAGn). For alanine-scanning substitutions,
nucleotide mutations with alanine conversions were introduced into native
hFasL/pCAGn using a site-directed mutagenesis kit (Stratagene). At amino
acid residue A240, a glycine was used for the substitution. Transient expression in COS cells was performed by using lipofectACE (Life Technologies, Rockville, MD) according to the manufacturer’s instruction. After 24 h of incubation, the culture medium was changed to ASF serum-free
medium (Ajinomoto, Tokyo, Japan). The culture supernatant was collected
after 72 additional hours, and the concentration of recombinant soluble
FasL was evaluated by ELISA using anti-FLAG mAb M2 as described
below.
ELISA for FasL/anti-FasL mAb binding and for FasL/Fas
binding
To evaluate the concentration of FLAG-tagged native or mutant FasL, an
Immulon 2 plate (Dynatech Laboratories, Chantilly, VA) was coated with
serially diluted native or mutant FasL for 16 h at 4°C. After washing with
0.05% Tween 20-PBS, the wells were blocked with 1.0% BSA-PBS for 2 h
at 37°C, 50 ␮l of anti-FLAG mAb (2 ␮g/ml) was added to each well, and
wells were incubated for 1 h at 37°C. Then, 50 ␮l of ⫻5000 diluted HRPconjugated goat anti-mouse Ig Ab (American Qualex, San Clemente, CA)
was added and incubated for 1 h at 37°C. The wells were developed with
100 ␮l 1 mg/ml orthophenylenediamine in 50 mM citrate-phosphate buffer
(pH 5.0) containing 0.03% H2O2 and stopped with 50 ␮l of 2 N H2SO4. OD
(A450 – 490) was measured on an automated ELISA reader (Molecular Devices, Sunnyvale, CA). Serial dilutions of native FasL, which was affinity
purified by a NOK2 column from the supernatant of native hFasL/pCAGn
cDNA-transfected COS cells, was used as the standard.
For evaluating binding of anti-FasL mAbs to native or mutant FasL, the
Immulon 2 plate was coated with native or mutant FasL (5 ␮g/ml) for 16 h
at 4°C. After blocking with 1.0% BSA-PBS, serially diluted NOK1, -2, and
-3 or humanized NOK2 (RNOK203) was added and incubated for 1 h at
37°C. Then HRP-conjugated goat anti-mouse Ig Ab and anti-human Ig Ab
(American Qualex) were used to detect the bound NOK1, -2, and -3 and
RNOK203, respectively.
For evaluating binding to Fas, the Immulon 2 plate was coated with 1
␮g/ml human Fas-human IgG1 Fc chimeric protein (Fas-Ig; Alexis, San
Diego, CA) for 16 h at 4°C. After blocking with 1.0% BSA-PBS, 500
ng/ml of native or mutant FasL was added to each well and incubated for
1 h at 37°C. The bound FasL was evaluated by subsequent incubations with
anti-FLAG mAb and HRP-labeled anti-mouse Ig Ab as described above.
Chemical cross-linking and Western blot analysis
The culture supernatants containing native or mutant FasL were concentrated ⬃40-fold by using a Centriprep-10 (Amicon, Beverly, MA). Ten
microliters of aliquot was diluted with 30 ␮l PBS and then incubated with
or without 0.15 mM DSS (Pierce) for 10 min at 4°C. Then the samples
were subjected to SDS-PAGE under reducing conditions, followed by
Western blot analysis using anti-FLAG mAb, goat HRP-labeled antimouse Ig Ab, and ECL (Amersham Pharmacia Biotech, Little Chalfont,
U.K.).
Cytotoxicity assay
Cytotoxic activity of native or mutant FasL was tested against hFas/
WR19L cells by the Alamar blue method according to the manufacturer’s
instruction (Alamar Biosciences, Sacramento, CA). Briefly, hFas/WR19L
(1 ⫻ 104) cells were incubated with serially diluted native or mutant FasL
in a total volume of 100 ␮l. After a 16-h incubation, 10 ␮l Alamar blue was
added to the culture, and the culture was further incubated for 4 h. Fluorescence of the reduced Alamar blue was detected on a Fluoroscan (Titertec Fluoroscan II; Labosystems, Tokyo Japan) at 590 nm by an excitation
at 544 nm.
Computer modeling of human FasL and Fas
A three-dimensional molecular model of human FasL monomer was built
by MODELER (Accelrys, San Diego, CA), a program that implements
comparative modeling by maximal satisfaction of spatial restraints. The
crystallographic structures of both TNF-␣ (12, 13) and LT-␣ (14)
(Brookhaven Protein Data Bank entries 1TNF and TNR) were used as the
templates. After molecular modeling of FasL monomer, three copies of
each monomer were superimposed on the structure of TNF trimer (1TNF).
The trimer structure was optimized by 100 steps of conjugate gradient
minimization using the CHARMm program (Accelrys). In a similar way,
molecular models of the FasL/Fas complex were predicted on the basis of
crystal structure of the human LT-␣-human TNFRI x-ray complex (19).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The cDNAs encoding murine Ig VH and VL regions were isolated by RTPCR from NOK1-, NOK2-, or NOK3-producing hybridoma. The VH region was amplified using AAG CTT GCC GCC ACC ATG GAA TGG
AGC TGG GTC TTT as the 5⬘ primer and GGA TCC ACT CAC CTG
AGG AGA CGG TGA as the 3⬘ primer. The VL region was amplified using
AAG CTT CGC CAC CAT GAA GTT GCC TGT TAG GCT G as the 5⬘
primer and GGA TCC ACT TAC GTT TTA TTT CCA GCT T as the 3⬘
primer. Each 5⬘ and 3⬘ primer was tagged with the HindIII and BamHI
sites, respectively. After subcloning of the cDNA into pBluescript II
SK(⫹) TA cloning vector (Stratagene, La Jolla, CA), the nucleotide sequence was determined by using an automated sequencer (Applied Biosystems, Foster City, CA) and the fluoresceinated dye terminator cycle
sequencing method. Humanization of NOK2 was performed essentially as
described previously (21, 22). Human VH cDNA (SGI, kindly provided by
Dr. M. M. Bendig, Medical Research Council Collaborative Center, London, U.K.) and VL cDNA from a human PBMC cDNA library were used
as the templates. In practice, most parts of complementarity-determining
regions (CDRs) of human VH and VL regions were genetically replaced
with the amino acid residues corresponding NOK2 CDRs by using a sitedirected mutagenesis kit (Stratagene) according to the manufacturer’s instruction. Some additional residues in human framework regions (FRs)
were also were genetically substituted by corresponding amino acid residues in the NOK2 FRs on the basis of our computed molecular models and
the knowledge from previous Ab-humanization studies. The HindIIIBamHI fragments of humanized NOK2 VH and VL cDNAs were then
transferred to HindIII-BamHI sites of pCAG-␥1 and pCAG-␬, respectively.
pCAG-␥1 is a mammalian expression vector modified from HCMVVH0.5␤-␥1 (23) in which the CMV promoter was replaced with the
chicken ␤-actin promoter and rabbit ␤-globin splicing acceptor. pCAG-␬
was generated by replacing the human ␥1 constant region in pCAG-␥1
with the ␬ constant region. CHO cells were transfected with both pCAG-␥1
and pCAG-␬ carrying humanized NOK2 VH and VL cDNA, respectively,
by electroporation. After selection with neomycin and methotrexate (both
from Sigma-Aldrich, St. Louis, MO), humanized and reshaped NOK2
mAbs were purified by using a protein G column from the culture supernatants. Among several versions of humanized NOK2 mAbs, one mAb,
designated RNOK203, was selected for its strong ability to inhibit FasL
cytotoxicity against hFas/WR19L.
3267
3268
FUNCTIONAL DETERMINANTS ON HUMAN FAS LIGAND
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FIGURE 1. Construction of humanized NOK2. A, Amino acid sequences of murine NOK1, -2, and -3 VH (upper) and VL (lower) regions. Amino acid
sequences of VH and VL regions of murine NOK1, -2, and -3 mAbs are aligned. The CDR1, -2, and -3 regions are boxed. The amino acid residues shared
among NOK1, -2, and -3 are indicated by an asterisk for a match of three of three and .a filled circle for a match of two of three. B, Amino acid sequences
The Journal of Immunology
3269
Table I. Effect of alanine substitutions on reactivity with anti-FasL mAbsa and FasL cytotoxicityb
FasL Mutants
NOK1
NOK2
NOK3
RNOK203
Cytotoxicity
⫺
⫺
0.876
0.147
⬎1.000
⬎1.000
⫺
0.000
⫺
0.107
⫺
⫺
⬎1.000
⫺
⬎1.000
⫺
0.652
⬎1.000
0.793
0.758
0.638
⬎1.000
0.379
0.713
0.028
0.145
0.439
0.817
⫺
⫺
⬎1.000
0.406
0.250
⬎1.000
0.964
⫺
⫺
⫺
0.851
0.800
⫺
0.247
0.840
⫺
⫺
⫺
⫺
0.000
0.035
⬎1.000
⬎1.000
⫺
⬎1.000
⫺
0.174
⫺
⫺
⬎1.000
⫺
⬎1.000
⫺
0.781
⬎1.000
⬎1.000
⬎1.000
0.809
⬎1.000
0.613
0.968
0.085
0.218
0.508
⬎1.000
⫺
⫺
⬎1.000
0.686
0.861
⬎1.000
0.370
⫺
⫺
⫺
⬎1.000
0.862
⫺
0.445
0.000
⫺
⫺
⫺
⫺
0.590
0.000
0.000
⬎1.000
⫺
0.000
⫺
0.000
⫺
⫺
⬎1.000
⫺
⬎1.000
⫺
0.350
0.802
0.604
⬎1.000
0.585
⬎1.000
0.602
⬎1.000
0.000
0.266
0.311
⬎1.000
⫺
⫺
⬎1.000
0.885
0.083
0.576
0.000
⫺
⫺
⫺
0.606
0.723
⫺
0.091
0.080
⫺
⫺
⫺
⫺
0.204
0.097
⬎1.000
⬎1.000
⫺
⬎1.000
⫺
0.100
⫺
⫺
⬎1.000
⫺
⬎1.000
⫺
0.697
⬎1.000
⬎1.000
⬎1.000
0.885
⬎1.000
0.558
0.768
0.068
0.175
0.411
0.842
⫺
⫺
⬎1.000
0.589
0.290
0.721
0.128
⫺
⫺
⫺
0.708
0.823
⫺
0.379
0.000
⫺
⫺
⫺
⫺
0.000
0.000
0.630
0.909
⫺
⬎1.000
⫺
⬎1.000
⫺
⫺
⬎1.000
⫺
0.000
⫺
0.000
0.407
0.579
0.000
0.917
0.880
0.000
1.000
0.000
0.000
0.000
0.550
⫺
⫺
⬎1.000
0.800
0.000
⬎1.000
0.000
⫺
⫺
⫺
⬎1.000
0.755
⫺
0.786
0.000
⫺
⫺
d
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
a
Relative binding of NOK1, -2, and -3 and RNOK203 to mutant FasL was evaluated by ELISA. Relative binding was calculated as follows:
concentration of native FasL (OD ⫽ 0.3)/concentration of mutant FasL (OD ⫽ 0.3). Similar results were obtained from three independent experiments.
b
Relative cytotoxic activity of mutant FasL was tested against hFas/WR19L cells in a 16-h Alamar blue assay. Relative cytotoxicity ⫽ ED50 of mutant
FasL/ED50 of native FasL. Similar results were obtained from three independent experiments.
c
The amino acid residues predicted to be exposed on the FasL trimer surface are indicated by circles.
d
⫺, Nonproductive.
Results and Discussion
Humanization of murine anti-human FasL mAb
To perform humanization, we first determined the amino acid sequences of VH and VL of murine anti-human FasL mAbs, NOK1,
NOK2, and NOK3, by sequencing RT-PCR-generated cDNAs
(Fig. 1A). Although the NOK1, -2, and -3 mAbs were derived from
the same mouse, their amino acid sequences in the VH and VL
CDR3 regions, which correspond to V(D)J junctions, were significantly different. In addition, V and J gene usages in NOK1, -2, and
-3 were also distinct as follows: NOK1, VH1/JH1 and V␬10/J␬1;
NOK2, VH1/JH4 and V␬1/J␬2; and NOK3, VH1/JH1 and V␬19/J␬1.
These differences suggest that NOK1, -2, and -3 were derived from
distinct B cell clones.
Because NOK2 neutralized FasL cytotoxicity more efficiently
than NOK1 and NOK3 (20), we subjected NOK2 to the humanization. Most parts of CDRs in the NOK2 VH and VL and some FR
amino acid residues were genetically transferred to the corresponding regions in the human VH and VL templates, respectively (Fig.
1B). A recombinant humanized NOK2, designated RNOK203, was
purified from culture supernatants of CHO cells transfected with
of murine NOK2 and humanized NOK2 (RNOK203) at VH (upper) and VL (lower) regions. The amino acid residues grafted from murine NOK2 are
indicated by asterisks. C, Neutralizing activity of RNOK203. Cytotoxic activity of native FasL (100 ng/ml) against hFas/WR19L cells was tested in the
presence of the indicated doses of NOK2 or RNOK203 in a 16-h Alamar blue assay. Data are indicated as mean ⫾ SD of triplicate samples. ⴱ, p ⬍ 0.05
compared with NOK2 (two-sample t test). Similar results were obtained from three independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Y196A
F197A
R198A
G199A
Q200A
S201A
C202A
N203A
N204A
L205A
P206A
L207A
S208A
H209A
K210A
V211A
Y212A
M213A
R214A
N215A
S216A
K217A
Y218A
P219A
Q220A
D221A
L222A
V223A
M224A
M225A
E226A
G227A
K228A
M229A
M230A
S231A
Y232A
C233A
T234A
T235A
G236A
Q237A
M238A
W239A
A240G
Surfacec
3270
both the humanized VH (␥1) cDNA and VL (␬) cDNA. As represented in Fig. 1C, RNOK203 neutralized FasL cytotoxicity more
efficiently than the original NOK2.
Some humanized or human-mouse chimera mAbs that can neutralize proinflammatory cytokines such as TNF-␣ have already
been successfully used in clinical trials (24 –26). We previously
demonstrated the therapeutic effects of neutralizing anti-FasL mAb
in murine model of hepatitis and GVHD (6, 7). The humanized
anti-FasL mAb (RNOK203) we generated here may be useful for
clinical application to human diseases.
Epitope mapping of anti-human FasL mAbs
FIGURE 2. Reactivity of NOK2 with native or mutant FasL. Dose-dependent binding of NOK2 to native (F) or mutant (E, Œ, ‚, and f) FasL
was evaluated by ELISA. Culture medium was used as a negative control
(䡺). Data are shown as the means of triplicated wells. SDs were ⬍5%
(data not shown). Similar results were obtained from three independent
experiments.
FIGURE 3. Effect of alanine substitutions on FasL trimer formation.
Native (left) or mutant (right) FasL treated with (⫹) or without (⫺) a
chemical cross-linker, DSS, was subjected to SDS-PAGE under reducing
conditions. After transfer to polyvinylidene difluoride membrane, FasL
proteins were detected by anti-FLAG M2 mAb. The bands representing
monomer, dimer, and trimer are indicated by arrows.
though we presently do not know precise reasons for the dramatic
reduction of FasL production by the alanine substitutions performed in this study, these substitutions may affect the intracellular
trafficking or posttranscriptional modification of FasL protein. Further studies are needed to address these possibilities.
We first evaluated the reactivity of NOK2 to each FasL mutant
by ELISA. As represented in Fig. 2, an alanine substitution at
Q220A, led to an almost complete loss of NOK2 reactivity. In
addition to the alanine substitution at Q220A, the loss of ⬎75%
reactivity with NOK2 was also found by alanine substitution of
R198, G199, L205, D221, and M238, whereas that of Y218, L222,
G227, M230, or Q237 led to ⬃35– 40% loss of NOK2 reactivity
(Fig. 2 and Table I). In a similar way, we also examined the binding of NOK1 and NOK3 (Table I). As was the case with NOK2,
widespread distributions of amino acid residues with the loss of
reactivity were also noted with NOK1 and NOK3, suggesting that
NOK1, -2, and -3 recognize conformational epitopes. Comparison
of each epitope for NOK1, -2, and -3 revealed a largely overlapped
FIGURE 4. Effect of alanine substitutions on FasL binding to Fas. Binding of native or mutant FasL to immobilized Fas-Ig was evaluated by
ELISA using anti-FLAG mAb for detection. Data are indicated as mean ⫾
SD of triplicate samples. Similar results were obtained from three independent experiments.
FIGURE 5. Alignment of TNF family molecules (A) and ribbon diagram of FasL monomer (B). A, The extracellular region of FasL was aligned with
the indicated TNF family members. The region subjected to alanine-scanning mutagenesis is boxed. The regions corresponding to ␤-strand A to H are
indicated below. Highly conserved amino acid residues among TNF family members are indicated in red (match of five of five) or blue (match of four of
five). B, The deduced regions for ␤-strand A to H are indicated as arrows in the ribbon diagram of FasL monomer.
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We previously demonstrated that NOK1, -2, and -3 mAbs inhibited the binding of soluble Fas-Ig to FasL transfectants (20). Our
preliminary epitope analysis by peptide mapping suggested that
these mAbs recognize amino acid residues present in the area from
Y196 to A240 in human FasL (data not shown). Thus, to identify
the amino acid residues critically involved in shaping the NOK1,
-2, and -3 epitopes, each amino acid residue from Y196 to A240
was subjected to alanine-scanning mutagenesis. The mutant FasL
molecules were constructed as a FLAG-tagged soluble form. Forty-five expression plasmids carrying native or mutant FasL cDNA
were transiently transfected into COS cells. Concentration of the
secreted FasL proteins was determined by ELISA using antiFLAG mAb. Although most of the mutant constructs as well as the
native one led to the production of ⬎1 ␮g/ml of soluble FasL in
the culture supernatant, the alanine-substitutions at Y196, F197,
C202, N204, P206, L207, H209, V221, M224, M225, S231, Y232,
C233, G236, W239, and A240 mostly abolished the FasL production in the supernatant (Table I). It has previously been reported
that some amino acid substitutions in human FasL residues resulted in a significant reduction of FasL protein production. For
example, amino acid replacements at N-glycosylation sites such as
D260 significantly reduced FasL protein production (27). Al-
FUNCTIONAL DETERMINANTS ON HUMAN FAS LIGAND
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FIGURE 5
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FUNCTIONAL DETERMINANTS ON HUMAN FAS LIGAND
distribution of the amino acid residues with remarkable loss of
reactivity. However, relative importance of each residue in shaping
the epitopes appeared to vary among NOK1, -2, and -3. For example, the alanine-substitution of N203 and K228 significantly
reduced FasL binding to NOK2 but not to NOK1 and NOK3 (Table I). These results indicated that the epitopes for NOK1, -2, and
-3 are similar but distinct. Consistent with this notion, our previous
study showed that NOK1, -2, and -3 mAbs only partially competed
with each other for their binding to FasL (20).
In a similar way, we also compared the residues important for
NOK2 and RNOK203 epitopes. The residues important for
RNOK203 reactivity were mostly overlapped with those for NOK2.
However, at positions 212, 222, 228, and 230, NOK2 exhibited significantly higher reactivity to alanine-substituted mutants than
RNOK203 (Table I). This difference may be responsible for the
higher neutralizing activity of RNOK203 than NOK2 observed in
Fig. 1C.
totoxicity (Table I). However, some other residues such as K210
and N215 were necessary for cytotoxicity but not for the NOK1,
-2, and -3 epitopes. As compared with the NOK1 epitope, the
NOK2 and NOK3 epitopes exhibited relatively higher similarity
with the distribution of amino acid residues crucial for the cytotoxicity (Table I).
Amino acid residues important for FasL cytotoxicity
As shown in Fig. 5A, the amino acid residues subjected to alanine
scan were predicted to locate on the region from the middle part of
the ␤-strand C to the end of the E-F loop in the FasL sequence.
Although the TNF family members exhibit only low sequence similarity, it is well known that their three-dimensional structures are
well conserved (12–18). As represented in Fig. 5B, like other
members of the TNF family including TNF-␣ (12, 13), LT-␣ (14),
CD40L (15), and TRAIL (16 –18), FasL monomer is deduced to
conform a ␤ strand jellyroll topology composed of two flat ␤
sheets. In the ribbon diagram, the region subjected to alanine scan
was extended from the top to bottom at one side of the FasL molecule (Fig. 5B). Then we deduced conformational positions of
these amino acid residues on FasL by using a space-filling comparative molecular model of FasL in trimer form. As represented
in Fig. 6A, the residues subjected to alanine scan are mainly located along a long groove at the FasL monomer-monomer interface. However, some hydrophobic amino acid residues such as
Y196 and F197 were deduced not to exist on the surface due to the
conformational folding of ␤ sheets (Table I). As described above,
we revealed the wide-spread distribution of amino acid residues
critical for NOK1, -2, and -3 binding (Fig. 2 and Table I), and we
next predicted the conformational epitopes for NOK1, -2, and -3
on the three-dimensional FasL model. As illustrated in Fig. 6,
B–D, each conformational epitope for NOK1, -2, and -3 on the
FasL trimer model could be classified into two clusters; one locates
around N205 near the top, and another locates around Q220 and
D221 corresponding to the edge of ␤-strand D (Fig. 5A). This
suggests that NOK1, -2 and -3 recognize a largely overlapped,
huge three-dimensional epitope composed of at least two spatially
distant clusters of amino acids. Consistent with this notion, a computed molecular model of NOK2 V regions revealed that the Agrecognition site of NOK2 has the almost same size as a circle with
a 17-Å radius that can cover all of the amino acid residues on the
upper and lower clusters on the FasL trimer (data not shown).
However, the reactivities of these mAbs to these clusters, especially to the upper cluster, appeared to be slightly different. For
example, R198 and N203 in the upper cluster were not required for
NOK1 and NOK2 binding, respectively (Table I and Fig. 6, B–D).
This suggests that the upper cluster might be mainly responsible
for the different specificities of these mAbs. As described above,
neutralizing activity of RNOK203 was significantly increased as
compared with original NOK2, possibly due to the increased reactivity to the amino acid residues at 212, 222, 228, and 230 (Fig.
1C and Table I). The conformational positions of these amino acid
residues were deduced to be located in the upper and lower clusters
(Fig. 6E), suggesting that the humanization of NOK2 increased the
affinity for both the upper and lower epitopes.
Possible mechanism for blockade of FasL/Fas interaction by
mAbs
We further deduced conformational locations of the amino acid
residues important for FasL cytotoxicity. As illustrated in the space
full-filling molecular model of FasL (Fig. 6F), the amino acid residues critical for FasL cytotoxicity are mostly located on the
groove at FasL/FasL interface. Like the epitope for NOK1, -2, and
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A previous study by Schneider et al. (27) using some human FasL
mutants demonstrated that the P206 and T218 residues in human
FasL were critically involved in the interaction with Fas receptor.
However, more details about the FasL/Fas interaction remain to be
determined. Thus, we next tested the cytotoxic activity of our
panel of FasL mutants. A mouse T lymphoma cell line-derived
human Fas cDNA transfectant, hFas/WR19L, which is highly sensitive to human FasL-induced apoptosis, was used to evaluate the
cytotoxic activity of the mutants. As reported previously (27), we
also found that the alanine substitution at T218 led to an almost
complete loss of the cytotoxicity (Table I). In addition, the alanine
substitution at R198, G199, K210, Y212, N215, Y218, Q220,
D221, L222, K228, M230, and M238 also mostly abolished FasL
cytotoxicity against hFas/WR19L (Table I).
It has been known that the trimeric structure of TNF family
ligands is required for execution of biological activity. Indeed, a
substitution of F275 to L275 in human FasL, which mimics the
loss-of-function mutation found in gld mice, has been reported to
lead to an inadequate oligomerization of FasL molecule with
significant loss of cytotoxic activity (27, 28). Thus, we next examined whether the alanine substitutions affected the homotrimeric
nature of FasL by Western blot analysis using anti-FLAG mAb for
detection. With a chemical cross-linker, DSS, which stabilizes the
FasL trimer complex, ⬃90- and 60-kDa bands representing FasL
trimer and dimer, respectively, were observed with native FasL
(Fig. 3). Similar levels of trimerization were also observed with all
the FasL mutants tested, as represented by L222A, V223A,
E226A, G227A, and K228A. Consistent with a previous report
(11), a 30-kDa band representing FasL monomer was dominant
without the DSS cross-linking (Fig. 3). In a similar way, we also
tested all the other FasL mutants for their trimer formation and
found no difference among native and mutant FasL (data not
shown).
We next analyzed binding of native and mutant FasL to Fas by
ELISA using Fas-Ig as a capture and anti-FLAG mAb as a detector. As represented in Fig. 4, all the alanine substitutions with
significant loss of cytotoxicity were accompanied with ⬎65% loss
of Fas binding as compared with native FasL. These results suggest that the reduced cytotoxicity of the FasL mutants was primarily due to the reduced binding to Fas receptor rather than inadequate trimer formation.
A comparison of amino acid residues critical for NOK1, -2, and
-3 binding and those critical for cytotoxicity revealed some extents
of similarity. For example, the G199A and Q220A substitutions
led to significant loss of reactivities with all mAbs and FasL cy-
Molecular modeling of conformational epitopes for anti-FasL
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FIGURE 6. Conformational mapping of critical residues for NOK1, -2, and -3 binding and cytotoxicity on molecular model of FasL. A, Residues
subjected to alanine-scanning mutagenesis are indicated as blue or green on the surfaces composed of two FasL monomers (left monomer, white and blue;
right monomer, pink and green) in a space-filling model of FasL trimer complex. B–F, Residues important for NOK1 (B), NOK2 (C), NOK3 (D), or
RNOK203 (E) binding or these important for FasL cytotoxicity (F) are indicated by different colors (red, ⬎75% loss of NOK1, -2, and -3 reactivity or FasL
cytotoxicity; green, 50 –75% loss; yellow, 25–50% loss; and blue, ⬍25% loss).
FUNCTIONAL DETERMINANTS ON HUMAN FAS LIGAND
-3 and RNOK203, these residues could be classified into two clusters, one around D221 near the bottom corresponding to the D-E
loop and ␤ strand E (Fig. 5, A and B) and another around K238
near the top. This suggests that the interaction with Fas takes place
at two distinct sites on FasL, the outer tip-forming D-E loop and the
region near the top of the FasL molecule. At these contact sites,
one FasL molecule binds two Fas molecules, which leads to the
aggregation of cytoplasmic death domains resulting in Fas-associated death domain recruitment. NOK1, -2, and -3 and RNOK203
seem to interrupt the FasL/Fas interaction by competing with Fas
at both of these FasL/Fas contact sites on FasL. However, the
lower cluster around Q220 important for FasL cytotoxicity was
relatively lager than those for NOK1, -2, and -3 and RNOK203
epitopes (Fig. 6, B–F). Although the upper cluster crucial for cytotoxicity was relatively well overlapped with those for NOK2 and
NOK1 epitopes, the upper cluster for NOK1 epitope appeared to
be shifted more closely to the top of FasL. These results suggest
that NOK1, -2, and -3 and RNOK203 might be so bulky that these
mAbs can inhibit the binding of Fas by directly or indirectly masking the Fas-binding sites on FasL.
Molecular modeling of FasL/Fas contact sites
Previous crystallographic studies of LT-␣/TNF-RI and TRAIL/
TRAILR2 complexes revealed that these ligand-receptor pairs interact with each other at two sites (18, 19). Also in our threedimensional model of the FasL/Fas complex, the trimerization of
FasL monomer forms three clefts at the interfaces between FasL
monomers (Fig. 7, A and B). It seems that three Fas molecules bind
diagonally along these clefts on the FasL trimer. Furthermore, one
Fas molecule binds two FasL molecules via two distinct contact
sites on FasL monomer at the outer tip-forming loop near the bottom and the side of cleft near the top (Fig. 7, A and B).
It is well known that not only the conformation of polypeptide
backbone but also the complementarity of the charge pattern and
hydrophobicity on the intermolecular surface determine the affinity
of a ligand-receptor pair. On the basis of conformational information, we finally deduced a more detailed molecular basis for the
interaction between FasL and Fas. In the upper cluster region (Fig.
7C), R198 on FasL ␤-strand C (Fig. 5A) was predicted to form a
hydrogen bond with D92 on the second CRD of Fas. Furthermore,
FIGURE 7. Molecular model of FasL/Fas interaction. Top (A) or side (B) view of the FasL/Fas complex in ribbon diagram. Each FasL and Fas molecule
in the trimer complex is indicated by different colors (FasL, cyan, green, and yellow; and Fas, orange, red, and purple). The predicted FasL/Fas interaction
sites are marked by circles. C and D, Close-up view of the FasL/Fas contact sites. The upper FasL/Fas contact sites around FasL R198 and Fas E93 (C)
and the lower contact site around FasL Y218 and Fas R86 (D) are illustrated as well-eyed stereo view.
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Acknowledgments
We sincerely thank Dr. S. Kazuno and K. Murayama for help.
References
1. Nagata, S. 1997. Apoptosis by death factor. Cell 88:355.
2. Suda, T., T. Takahashi, P. Golstein, and S. Nagata. 1993. Molecular cloning and
expression of the Fas ligand, a novel member of the tumor necrosis factor family.
Cell 75:1169.
3. Bellgrau, D., D. Gold, H. Selawry, J. Moore, A. Franzusoff, and R. C. Duke.
1995. A role for CD95 ligand in preventing graft rejection. Nature 377:630.
4. Griffith, T. S., T. Brunner, S. M. Fletcher, D. R. Green, and T. A. Ferguson. 1995.
Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270:
1189.
5. Kondo, T., T. Suda, H. Fukuyama, M. Adachi, and S. Nagata. 1997. Essential
roles of the Fas ligand in the development of hepatitis. Nat. Med. 3:409.
6. Seino, K., N. Kayagaki, K. Takeda, K. Fukao, K. Okumura, and H. Yagita. 1997.
Contribution of Fas ligand to T cell-mediated hepatic injury in mice. Gastroenterology 113:1315.
7. Hattori, K., T. Hirano, H. Miyajima, N. Yamakawa, M. Tateno, K. Oshimi,
N. Kayagaki, H. Yagita, and K. Okumura. 1998. Differential effects of anti-Fas
ligand and anti-tumor necrosis factor ␣ antibodies on acute graft-versus-host
disease pathologies. Blood 91:4051.
8. Locksley, R. M., N. Killeen, and M. J. Lenardo. 2001. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487.
9. Screaton, G., and X. N. Xu. 2000. T cell life and death signalling via TNFreceptor family members. Curr. Opin. Immunol. 12:316.
10. Smith, C. A., T. Farrah, and R. G. Goodwin. 1994. The TNF receptor superfamily
of cellular and viral proteins: activation, costimulation, and death. Cell 76:959.
11. Suda, T., and S. Nagata. 1994. Purification and characterization of the Fas-ligand
that induces apoptosis. J. Exp. Med. 179:873.
12. Jones, E. Y., D. I. Stuart, and N. P. Walker. 1989. Structure of tumor necrosis
factor. Nature 338:225.
13. Eck, M. J., and S. R. Sprang. 1989. The structure of tumor necrosis factor-␣ at
2.6-Å resolution: implications for receptor binding. J. Biol. Chem. 264:17595.
14. Eck, M. J., M. Ultsch, E. Rinderknecht, A. M. de Vos, and S. R. Sprang. 1992.
The structure of human lymphotoxin (tumor necrosis factor-␤) at 1.9-Å resolution. J. Biol. Chem. 267:2119.
15. Karpusas, M., Y. M. Hsu, J. H. Wang, J. Thompson, S. Lederman, L. Chess, and
D. Thomas. 1995. A crystal structure of an extracellular fragment of human
CD40 ligand. Structure 3:1031.
16. Cha, S. S., M. S. Kim, Y. H. Choi, B. J. Sung, N. K. Shin, H. C. Shin, Y. C. Sung,
and B. H. Oh. 1999. 2.8-Å resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity. Immunity 11:253.
17. Cha, S. S., H. C. Shin, K. Y. Choi, and B. H. Oh. 1999. Expression, purification
and crystallization of recombinant human TRAIL. Acta Crystallogr. D Biol.
Crystallogr. 55:1101.
18. Hymowitz, S. G., H. W. Christinger, G. Fuh, M. Ultsch, M. O’Connell,
R. F. Kelley, A. Ashkenazi, and A. M. de Vos. 1999. Triggering cell death: the
crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol. Cell
4:563.
19. Banner, D. W., A. D’Arcy, W. Janes, R. Gentz, H. J. Schoenfeld, C. Broger,
H. Loetscher, and W. Lesslauer. 1993. Crystal structure of the soluble human 55
kd TNF receptor-human TNF ␤ complex: implications for TNF receptor activation. Cell 73:431.
20. Kayagaki, N., A. Kawasaki, T. Ebata, H. Ohmoto, S. Ikeda, S. Inoue, K. Yoshino,
K. Okumura, and H. Yagita. 1995. Metalloproteinase-mediated release of human
Fas ligand. J. Exp. Med. 182:1777.
21. Foote, J., and G. Winter. 1992. Antibody framework residues affecting the conformation of the hypervariable loops. J. Mol. Biol. 224:487.
22. Winter, G., and C. Milstein. 1991. Man-made antibodies. Nature 349:293.
23. Maeda, H., S. Matsushita, Y. Eda, K. Kimachi, S. Tokiyoshi, and M. M. Bendig.
1991. Construction of reshaped human antibodies with HIV-neutralizing activity.
Hum. Antibodies Hybridomas 2:124.
24. Clark, M. 2000. Antibody humanization: a case of the “Emperor’s new clothes”?
Immunol. Today 21:397.
25. Elliott, M. J., R. N. Maini, M. Feldmann, A. Long-Fox, P. Charles, P. Katsikis,
F. M. Brennan, J. Walker, H. Bijl, J. Ghrayeb, et al. 1993. Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor ␣.
Arthritis Rheum. 36:1681.
26. Elliott, M. J., R. N. Maini, M. Feldmann, J. R. Kalden, C. Antoni, J. S. Smolen,
B. Leeb, F. C. Breedveld, J. D. Macfarlane, H. Bijl, et al. 1994. Randomised
double-blind comparison of chimeric monoclonal antibody to tumor necrosis factor ␣ (cA2) versus placebo in rheumatoid arthritis. Lancet 344:1105.
27. Schneider, P., J. L. Bodmer, N. Holler, C. Mattmann, P. Scuderi, A. Terskikh,
M. C. Peitsch, and J. Tschopp. 1997. Characterization of Fas (Apo-1, CD95)-Fas
ligand interaction. J. Biol. Chem. 272:18827.
28. Hahne, M., M. C. Peitsch, M. Irmler, M. Schroter, B. Lowin, M. Rousseau,
C. Bron, T. Renno, L. French, and J. Tschopp. 1995. Characterization of the
non-functional Fas ligand of gld mice. Int. Immunol. 7:1381.
29. Goh, C. R., and A. G. Porter. 1991. Structural and functional domains in human
tumour necrosis factors. Protein Eng. 4:385.
30. Van Ostade, X., J. Tavernier, T. Prange, and W. Fiers. 1991. Localization of the
active site of human tumour necrosis factor (hTNF) by mutational analysis.
EMBO J. 10:827.
31. Starling, G. C., J. Bajorath, J. Emswiler, J. A. Ledbetter, A. Aruffo, and
P. A. Kiener. 1997. Identification of amino acid residues important for ligand
binding to Fas. J. Exp. Med. 185:1487.
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FasL G199 was expected to interact with Fas E93 and G94 via van
der Waals force. In the lower contact region, Y218 on the FasL D-E
loop that is conserved among death-inducing TNF family members
(Fig. 5A) has been reported to be important for LT-␣ and TNF-␣
binding to TNFRI (19, 29, 30). In addition, R86 on the second
CRD of Fas has been shown to be a critical residue for direct
interaction with FasL Y218 (31). In agreement with these notions,
FasL Y218 was predicted to directly bind to Fas R86 by forming
a hydrogen bond in our molecular model (Fig. 7D). Furthermore,
our model suggested that Fas R86 also interacts with FasL Q220
and D221 on ␤-strand E, forming a network of hydrogen bonds
and van der Waals contact.
In conclusion, we revealed that the anti-human FasL mAbs inhibit the cytotoxic activity of FasL by masking both of two distinct
Fas binding sites on FasL. Our molecular model of the FasL/Fas
interaction would facilitate the drug design for interrupting the
FasL/Fas interaction. In addition, the humanized NOK2 we generated in this study would be useful for clinical application to
human diseases in which the FasL/Fas system plays a key role in
the pathogenesis.
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