of June 13, 2013.
This information is current as
Immunotoxins for the Treatment of Cancer
Pseudomonas Exotoxin (PE38) Used to Make
Associated with a Truncated Form of
Characterization of the B Cell Epitopes
Robert J. Kreitman, Raffit Hassan and Ira Pastan
Byungkook Lee, Michihiro Nakamura, Jaulang Hwang,
Richard Beers, Robert J. Fisher, James J. Vincent,
Masanori Onda, Satoshi Nagata, David J. FitzGerald,
2006; 177:8822-8834; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Copyright © 2006 by The American Association of
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The Journal of Immunology
by guest on June 13, 2013
Characterization of the B Cell Epitopes Associated with a
Truncated Form of Pseudomonas Exotoxin (PE38) Used to
Make Immunotoxins for the Treatment of Cancer Patients1
Masanori Onda,2* Satoshi Nagata,2* David J. FitzGerald,* Richard Beers,* Robert J. Fisher,†
James J. Vincent,* Byungkook Lee,* Michihiro Nakamura,* Jaulang Hwang,*
Robert J. Kreitman,* Raffit Hassan,* and Ira Pastan3*
Recombinant immunotoxins composed of an Ab Fv fragment joined to a truncated portion of Pseudomonas exotoxin A (termed
PE38) have been evaluated in clinical trials for the treatment of various human cancers. Immunotoxin therapy is very effective in
hairy cell leukemia and also has activity in other hemological malignancies; however, a neutralizing Ab response to PE38 in
patients with solid tumors prevents repeated treatments to maximize the benefit. In this study, we analyze the murine Ab response
as a model to study the B cell epitopes associated with PE38. Sixty distinct mAbs to PE38 were characterized. Mutual competitive
binding of the mAbs indicated the presence of 7 major epitope groups and 13 subgroups. The competition pattern indicated that
the epitopes are discrete and could not be reproduced using a computer simulation program that created epitopes out of random
surface residues on PE38. Using sera from immunotoxin-treated patients, the formation of human Abs to each of the topographical
epitopes was demonstrated. One epitope subgroup, E1a, was identified as the principal neutralizing epitope. The location of each
epitope on PE38 was determined by preparing 41 mutants of PE38 in which bulky surface residues were mutated to either alanine
or glycine. All 7 major epitope groups and 9 of 13 epitope subgroups were identified by 14 different mutants and these retained
high cytotoxic activity. Our results indicate that a relatively small number of discrete immunogenic sites are associated with PE38,
most of which can be eliminated by point mutations. The Journal of Immunology, 2006, 177: 8822–8834.
small m.w. compounds, proteins are usually large enough to in-
duce an immune response in patients (3). Not surprisingly, proteins
obtained from nonmammalian sources induce Abs more frequently
than proteins of human origin (3–5).
Our laboratory is developing recombinant immunotoxins for the
treatment of cancer (6–13). These agents are composed of a 38-
kDa portion of Pseudomonas exotoxin A (PE38),4which is of
bacterial origin, and the Fv portion of a mAb genetically fused to
ver the past 20 years, a variety of protein therapeutics
has been administered to humans including mAbs, pro-
tein growth factors, and enzymes (1, 2). Compared with
it. The binding activity of the Fv moiety targets the immunotoxin
to Ag-positive cells which are killed by the cytotoxic activity of
the toxin moiety (11, 13). When immunotoxins are administered to
patients, neutralizing Abs often develop within 3 wk. These Abs,
which almost always react with PE38 and very infrequently with
the Fv, limit the number of treatment cycles that can be given (8,
14, 15). Fortunately, patients with certain leukemias and lympho-
mas, including hairy cell leukemia, infrequently make Abs to the
immunotoxin and can receive the benefit of many cycles (9, 12).
This nonresponsiveness probably results from damage to the im-
mune system, either due to previous chemotherapy or because the
leukemia causes immune suppression. Consequently, we have had
our greatest successes when treating patients with leukemia. Over
half of the patients with life-threatening drug-resistant hairy
cell leukemia achieved a complete remission after receiving
three or more cycles of the immunotoxin BL22 targeted to
CD22 (9, 12). This result suggests that immunotoxin therapy is
more likely to be successful if multiple cycles of treatment can
Several approaches have been proposed to decrease the immuno-
genicity of foreign proteins such as PE38. One involves shielding the
protein from the immune system by conjugating high m.w. polyeth-
ylene glycol (PEG) to the immunotoxin (16, 17). This approach re-
quires attaching the PEG at locations on the toxin that do not interfere
with its cytotoxic activity (18). Another approach is to identify and
remove epitopes of T cells (19, 20) or of B cells (21–23) by site-
directed mutagenesis. T cell epitopes recognized by Th cells are com-
plexes of the peptide fragments derived from the Ag and the MHC
in humans (25) and because T cells stimulated by one epitope can
stimulate responses to different B cell epitopes on the same Ag (26,
27), it is a formidable task to remove all possible T cell epitopes from
*Laboratory of Molecular Biology, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892; and†Protein Chemistry
Laboratory, Research Technology Program, Science Applications International Cor-
poration, National Cancer Institute, Frederick, MD 21702
Received for publication March 16, 2006. Accepted for publication September
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.
1This work was supported in part by the Intramural Research Program of the Center
for Cancer Research, National Cancer Institute, National Institutes of Health (NIH),
and was additionally funded in part with federal funds from the National Cancer
Institute, National Institutes of Health, under contract N01-CO-12400. The content of
this publication does not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names, commercial products,
or organizations imply endorsement by the U.S. government.
2M.O. and S.N. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Ira Pastan, Laboratory of Mo-
lecular Biology, National Cancer Institute, 37 Convent Drive, Room 5106, Bethesda,
MD 20892-4264. E-mail address: email@example.com
4Abbreviations used in this paper: PE38, a 38-kDa portion of Pseudomonas exotoxin
A; PEG, polyethylene glycol; HFc, human immunoglobulin Fc fragment; RFc, rabbit
Fc; ICC-ELISA, immune complex capture-ELISA; ROC, receiver operating charac-
teristic; TMB, tetramethylbenzidine; SI, stability index.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
by guest on June 13, 2013
a foreign protein. Pretreating patients with immunosuppressive agents
has also been proposed as a way to reduce Ab responses but, to date,
very little progress has been reported (15). However, patients receiv-
ing anti-B cell chemotherapy such as cladribine may exhibit long
delays in responding to foreign proteins (28).
This study focuses on a strategy to identify the B cell epitopes
on PE38, with the ultimate goal of removing or modifying the most
prominent ones. The properties of B cell epitopes have not been as
well-characterized as those of T cell epitopes. Although some in-
vestigators have proposed that any region on the surface of a pro-
tein can be an epitope (29–33), most studies have reported the
existence of clusters of Ab epitopes on the Ag surface (34, 35).
Examination of the interfaces of the crystal structures of Ag-Ab
complexes have not yet identified a universal mode of interaction
or identified specific amino acids that are uniformly important in
these interactions (33, 36–40) although some prediction methods
have successfully identified highly immunogenic regions (41–43).
For proteins from related species, there are regions that are not
particularly immunogenic because paralog proteins have “edu-
cated” the immune system (44). But for a foreign protein such as
PE38, the presence of B cell epitope hot spots has not been dem-
onstrated. To address the issue of PE38 immunogenicity, we de-
cided it was first necessary to locate all detectable epitopes and
then evaluate their distribution for evidence of hot spots. We also
considered that some Ab-PE38 interactions would neutralize the
toxin and some would not.
A common approach for identifying immunogenic hot spots is
to use small fragments or peptides derived from a protein. We
previously used this approach and identified some epitopes on
PE38 (14, 45). However, this approach cannot locate discontinu-
ous conformational B cell epitopes (46–48). Conformational
epitopes can be located by evaluating loss of Ab reactivity with a
series of point mutants of the Ag (49, 50). Furthermore, because
each mutant should eliminate a single epitope, a series of mAbs
against individual epitopes is required to distinguish each epitope
from the others.
Because there is evidence that human and mouse Abs recognize
the same epitopes on foreign proteins (51–54), we made a large
panel of mouse anti-PE38 mAbs, and used these to determine the
number and location of the epitopes. We also demonstrated that
Abs to these epitopes are also present in human sera from immu-
Materials and Methods
The immunotoxins and their recombinant target proteins
Several PE38-based immunotoxins were used in this study. They are BL22
(9), LMB-2 (55), M1 (56), LMB-9 (57), SS1P (10), and T6 (58) and their
inactive mutants (59, 60). We also made both domains of PE38 (domain II
and III) separately (61). Table I summarizes the composition of these im-
munotoxins and their targets on cancer cells. All the immunotoxins and
domains were made by a standard protocol established in our laboratory
(63). In brief, the components of immunotoxins were expressed in Esch-
erichia coli BL21 (?DE3) under a T7 promoter and harvested as inclu-
sion bodies. The protein was solubilized in 6 M guanidine hydrochlo-
ride under reducing conditions, and then refolded by dilution into a
refolding solution containing redox shuffling reagents. Active mono-
meric protein was purified by ion exchange and size exclusion chro-
matography to near homogeneity. Protein concentrations were deter-
mined by a Bradford assay (Coomassie Plus; Pierce) using BSA as the
The extracellular domains of the target membrane proteins of the
immunotoxins (CD22, CD25, mesothelin, and CD30) were produced as
human Ig Fc fragment (HFc)-fusion proteins or rabbit Fc (RFc)-fusion
proteins in 293T cells. The cells were transiently transfected with the
corresponding pcDNA3-based plasmids and the Fc-fusion proteins were
harvested in the culture supernatants (64, 65). The Fc-fusion proteins
were purified by protein A-Sepharose (Amersham Biosciences) as de-
scribed previously (64).
Passive adsorption of proteins onto plastic surfaces often alters the protein
conformation by destroying conformational epitopes or revealing cryptic
ones (66–68). To avoid these potential problems we have devised an in-
direct ELISA termed an immune complex capture ELISA (ICC-ELISA).
The ICC-ELISA detects Ag-Ab reactions that occur in solution. In this
ELISA, microtiter plates (MaxiSorp; Nalge Nunc) were coated with 100
ng/50 ?l/well CD22-HFc, CD25-RFc, or CD30-HFc in PBS overnight at
4°C. In separate tubes, the anti-PE38 mAb samples diluted in blocking
buffer (25% DMEM, 5% FBS, 25 mM HEPES, 0.5% BSA, 0.1% sodium
azide in PBS) were mixed with 2 ?g/ml of an immunotoxin containing
PE38 fused with an Fv reactive for CD22, CD25, or CD30. After washing
the plates with PBS containing 0.05% Tween 20, the immunotoxin-Ab
mixtures were transferred to each well (50 ?l/well). The amount of
immune complex captured by the Fc fusion proteins was detected by
HRP-conjugated goat anti-mouse IgG (H?L; no. 115-035-146; Jackson
ImmunoResearch Laboratories) or HRP-rat anti-mouse ? mAb (no. 04-
6620; Zymed Laboratories) followed by tetramethylbenzidine (TMB)
substrate kit (Pierce).
Production of mAbs
Immunization of mice with immunotoxins was conducted under various
conditions to increase the chances of obtaining mAbs to all possible
Table I. Properties of PE38-based immunotoxins used in this study
Toxin portion Activitya
Ref. Target Formatb
aSpecific cytotoxicity was determined using the cells expressing target Ags as described in the references.
bscFv, Single-chain Fv fragment; dsFv, disulfide-bond stabilized Fv fragment.
cPE38 contains amino acids 251–364 and 381–613 of Pseudomonas exotoxin A. Numbering is the same as that used to
describe the crystal structure (Protein Data Bank, 1IKQ) (62).
dM1-scFv was derived from LMB-2 scFv and includes nine mutated residues to lower the isoelectric point (56).
eE553 is the NAD binding site and R276 is one of the basic amino acids that constitute the furin cleavage site (59, 60). Both sites
are essential for cytotoxicity (11). Point mutations of either residue are known to reduce the PE activity to nondetectable levels.
fMaltose binding protein fused with aa 251–364 of Pseudomonas exotoxin A.
8823The Journal of Immunology
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epitopes. The immunogen and immunization conditions are summarized in
a part of Table II and Results. All the animal protocols were approved by
the Animal Care and Use Committee at the National Institutes of Health.
Hybridomas were produced by a cell fusion protocol using SP2/0-neo cells
(69). The ICC-ELISA was used to select 60 mAbs (57 mAbs from this
study and three previous mAbs in Ref. 70) that bind to PE38 in solution.
Ig isotypes were determined by mouse mAb isotyping reagents (ISO2;
Sigma-Aldrich). IgG concentrations in the culture supernatants were de-
termined by a sandwich ELISA using isotype-matched IgG controls (no.
90-6551, mouse Ig panel; Zymed Laboratories) as previously described
(71). In some experiments, mAbs were purified using protein G Sepharose
Topographical epitope mapping
The classification of the mAbs into epitope groups was conducted by the
mutual competition of all possible pairs of the anti-PE38 mAbs (60 ? 60 ?
3600) in a label-free competitive ELISA (72). For example: an indicator mAb
1 was captured by goat anti-mouse IgG Fc that had been coated on the
microtiter plates. In a separate tube, an excess amount of competitor mAb 2
was diluted in the blocking buffer and mixed with an appropriate concentration
of T6 immunotoxin (anti-CD30) overnight at 4°C. The plates were washed
twice and the mixtures in the tubes were transferred to each well. The immune
complexes captured on plates were probed with CD30-HFc followed by
HRP-conjugated goat anti-human IgG and TMB substrate.
The binding of mAb 1 to the mAb 2-T6 immune complex was deter-
mined as the percentage of the binding without mAb 2. As a result, each
mAb 1 had a set of normalized competition indexes for each mAb 2-Ag
complex. The pairwise matrix table of the competition indexes was ana-
lyzed by cluster analysis to group the mAbs based on the topographical
relationship of their epitopes (72).
In some experiments, patient sera containing human anti-PE38 Abs
were examined in a similar competition assay. The human serum samples
were obtained before and after patients with solid tumors received treat-
ment with the immunotoxins LMB-9 or SS1P during phase I clinical trials
(National Cancer Institute (NCI) protocol IDs: NCI-431 and NCI-01-C-
0011) (10, 57). These studies were approved by the Institutional Review
Board of the National Cancer Institute and all the patients gave written
informed consent. In the analysis, a 200-fold dilution of the human serum
in the blocking buffer was used in place of the competitor mAb to test their
binding to each epitope recognized by each capture mAb. M1 immunotoxin
(anti-CD25), CD25-RFc, and HRP-conjugated goat anti-rabbit IgG (Jack-
son ImmunoResearch Laboratories) were used for these experiments.
The binding kinetics of each mAb with PE38 was determined using a
Biacore biosensor (Biacore International) as previously described (73). In-
dividual mAbs were captured by rabbit anti-mouse Fc Ab that had been
covalently coupled to the sensor chip (CM5; Biacore). To detect interac-
tions with PE38, solutions of the immunotoxin BL22 were passed over the
mAbs. Sensorgrams were analyzed by simple Langmuir-binding isotherms.
In separate experiments, multisite binding assays were performed by
sequentially flowing different mAbs (nos. 36, 4, 21, 69, and 40) over SS1P
immunotoxin that had been immobilized on the sensor surface (74). Five
hundred resonance units of SS1P were immobilized on the chip, CM5. The
kinetics of mAb binding was measured in PBS by serially injecting 50
?g/ml of each mAb or a mixture over the chip.
Simulation analysis of the epitope location
To determine whether the epitope clusters can be reproduced by random
selection of epitopes from the surface of PE38, a computer-based simula-
tion analysis was conducted. A PE38 model was made by extracting res-
idues present in PE38 (251–364 and 381–605) from the crystal structure of
PE (62). Artificial epitopes were randomly created on the surface of the
model and their overlap was calculated as the Ab competition value in a
matrix. The number and quality of the artificial clusters were compared
with that from the experimental data. To create artificial epitopes on PE38,
all the exposed residues were selected from the PE38 model by Lee and
Richards’ method (75); artificial epitopes around the selected residues were
created by collecting surface exposed atoms around the central residue
incrementally until the outermost ring contained 60 or more nonhydrogen
atoms. The number 60 is the average number of atoms in the periphery of
the interface between four different Abs and the hen egg white lysozyme in
their crystal structures. Two artificial epitopes were considered to overlap
if they shared one or more atoms. Competition binding values for pairs of
virtual Abs to these epitopes were defined as one if there was any overlap,
zero otherwise. Ten competition-binding data sets for the evaluation were
generated by selecting the exposed residues randomly.
All sets of competition values, both artificial and experimental, were
clustered using the PyCluster software library developed by M. Eisen
htm#pycluster?). For comparison, each of the experimental competition in-
dexes (see above) was converted to either one or zero using 50% compe-
tition as the cutoff. The resulting matrix was then clustered. To evaluate the
quality of each clustering, we calculated the following values (true positive
rate and false positive rate) for each cluster number in the clustering and
plotted as a receiver operating characteristic (ROC) curve (76).
True positive rate (y-axis) ? filled elements inside clusters/elements
False positive rate (x-axis) ? filled elements outside clusters/elements
Filled elements ? empty elements ? number of mAbs2(60 ?
60 ? 3600)
Elements inside clusters ? elements outside clusters ? number of
mAbs2(60 ? 60 ? 3600)
An element ij is filled if Abs i and j compete (competition value ? 1),
empty if they do not.
Neutralization assay for the mAbs
For the neutralization assay, 6 ?g/ml (40 nM) of each mAb was incubated
with 5 ng/ml (80 pM) M1 immunotoxin for 30 min at 37°C and then
applied to Atac4 cells (A431 stably transfected with CD25) (55). After
24 h, incorporation of tritium-labeled leucine into cellular protein was mea-
sured as described previously (63). The neutralizing activity of each mAb
was expressed as the percent recovery of leucine incorporation (counts)
relative to the specific reduction by the immunotoxin without a mAb.
The association of the characteristics of the mAbs (neutralization activity,
association rate constant, dissociation rate constant, and affinity constant)
with the topographical epitopes was evaluated by a nonparametric statistics
(Mann-Whitney U test). mAbs assigned to each epitope were tested against
the other mAbs assigned to different epitopes with the null hypothesis that
the two populations of mAbs have identical distributions with the testing
characteristics (p ? 0.01). Ep2a was not evaluated because only one mAb
was assigned to this epitope.
Point mutants of PE38 used to locate each epitope
To locate specific epitopes, we produced a series of single point mutants
of PE38. We chose highly exposed surface residues (?70 Å2) as the
first candidates. Of 347 residues, there are 98 (28%) that exhibit an
average exposure of 113.1 ? 33.3 Å2covering 67% of the total surface
area. Because an epitope usually spans 6–8 aa and occupies 400–900
Å2(36, 37, 77), a relatively small number of mutants should be suffi-
cient to “cover” the entire surface of PE38 if they are evenly distributed.
We took advantage of our previous knowledge that mutations of either
R or E, followed by D, Q, or N tended to retain toxin function (18, 78).
the ICC-ELISA. Immunotoxins, comprising the 38-kDa portion (domain II
and III) of Pseudomonas exotoxin A fused to an Fv specific for target Ags
on cancer cells, are incubated with candidate mAbs. Because the reaction
takes place in solution, all epitopes on native PE38 are theoretically avail-
able. Complexes are captured to the plate with immobilized target ligands
such as CD22-Fc or similar molecules. The primary Ab is then detected
with an HRP-labeled secondary Ab.
ELISA to detect Abs reactive for soluble PE38. Principle of
8824B CELL EPITOPES OF PE38
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Table II. A variety of mAbs to the surface of PE38 produced in 10 different fusions
Number of mAbs Obtaineda
M1 ? 4
E553D ? 7
M1 ? 6
R276G ? 6
R276G ? 4
M1 ? 5
M1 ? 5
E553D ? 4
M1 ? 3 ? E553D ? 2
M1 ? 3 ? E553D ? 2
aAll mAbs were IgG1(? ? ?1) except for no. 16(? ? ?2a), no. 36(? ? ?2b), no. 37(? ? ?2b), and no. 49(? ? ?2b).
bIdentified by the mutual competition of mAbs to PE38 (Fig. 2).
cMeasured by a Biacore analysis using conditions where both paratopes and epitopes were in a native conformation (Fig. 7).
dImmunogen (shown in Table I) used except for the final boosts before the fusion. For example, “M1 ? 3 ? E553D ? 2” indicates three immunizations with M1 immunotoxin followed by two immunizations with LMB2-E553D. The injection
intervals were typically 2 wk apart. The injections were performed i.p., s.c., or i.v. Doses were 5–50 ?g/mouse. No adjuvant was used except for mouse no. 6, which received CFA in the first immunization.
eImmunogen used in the final boost 3 days before fusion, 10–50 ?g of the Ags were injected i.p. in PBS.
fThe affinities of mAbs no. 14 (Fusion 1, Ep4b) and no. 49 (Fusion 9, Ep4a) were not measured. mAb no. 57 (Fusion4, Ep1a) was excluded because of the exceptionally high-affinity constant (5.3 ? 1011M?1).
gThese mAbs were previously obtained (70).
8825The Journal of Immunology
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notoxin containing PE38 in the presence of 600-fold excess amounts of competitors (listed in columns). Strengths of competition are shown as percentages
in each box, which are shaded according to the key at the top of the figure. The competitive patterns were analyzed by a cluster analysis (shown in Fig.
3A). The number of epitope groups and subgroups was determined based on the SI as shown in Fig. 3B. Seven epitopes and 13 epitope subgroups are
separated by blue and green lines, respectively.
Topographical epitope mapping of mAbs by mutual competition. The binding of indicator mAbs (listed in rows) to anti-CD30 T6 immu-
8826B CELL EPITOPES OF PE38
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In addition, these residues often supply significant energy to Ag-Ab
binding through polar interactions or hydrogen bonds (36, 39, 40). We
included four mutants in the panel where residues had ?70 Å2in area.
The resulting mutant panel consists of only 41 mutants (12% of total
residues) but covers 28.3% (4,693/16,560 Å2) of the PE38 surface area.
The distances of the ? carbons of each residue to its nearest neighbor
(7.4 ? 3.0 Å) are close to the distances between all the exposed (?70
Å2) residues (5.0 ? 1.7 Å). To evaluate the side-chain contributions of
Ab binding to PE38, the mutated residues were replaced with A or G
except for E553D. The mutations in PE38 portion were introduced in
the anti-CD22 immunotoxin, BL22, except for R276G that was in the
anti-CD25 immunotoxin, LMB2. The point-mutant panel consists of
P268A, R276G, E282A, E285A, P290A, A300G, R302A, Q310A,
R313A, P319G, D324A, E327A, E331A, Q332A, E348A, R352A,
Q353A, D403A, R412A, N416A, E420A, R427A, E430A, E431A,
R432G, R458A, R467A, Q485A, R490A, R505A, R513A, L516A,
R529A, R538A, E548A, R551A, E553D, R576A, K590A, L597A, and
D599A. The mutant proteins were expressed and purified to ?95%
homogeneity by established protocols (63). All mutant proteins were
obtained with reasonable yields, usually 2–10% of starting material
(data not shown).
Competition assay to measure the reactivity of mAbs to PE38
Each PE38 point mutant was tested for its reactivity with each mAb in
solution to keep both Ag and Ab native during the reaction. We used 40
representative mAbs from the 60 mAbs in the competition assay to cover
all 7 major epitope groups and 12 of the 13 subgroups (Ep2a was excluded
because it contained only one mAb). To standardize the different affinity of
individual mAbs assigned to each epitope, the result was evaluated as
residual reactivity, which was defined as the ratio of the concentrations of
each mutant and of the wild type that were required for the binding to the
same amount of each mAb (79, 80). These values are close to the affinity
ratios as will be described in Results. In the assay, mAbs that failed to bind
mutant versions of PE38 were selectively captured by the antimesothelin
immunotoxin, SS1P, which had been indirectly coated on the plates via
mesothelin-Fc fusion proteins.
In brief, microtiter plates were coated with 100 ng/50 ?l/well me-
sothlin-RFc, followed by a 2-h incubation with 200 ng/100 ?l/well
SS1P. In separate tubes, a series of 4-fold dilutions of each mutant or
wild-type immunotoxin (0.04–10,000 ng/ml) were mixed with an ap-
propriate concentration of each mAb in blocking buffer at 4°C over-
night. The concentration of each mAb in the mixtures had been prede-
termined as a concentration that gave a half maximum signal in this
ELISA. After washing the plates, the immunotoxin-mAb mixtures in
the tubes were transferred to each well (50 ?l/well). The uncomplexed
mAb in the mixtures was captured by the SS1P coated on the plate
during a 1-h incubation. The mAbs were finally detected by HRP-con-
jugated goat anti-mouse IgG (H?L), followed by TMB substrate. The
concentrations of each mutant that reduced the signal by 50% (IC50)
were calculated by fitting to a four-parameter logistic curve. The resid-
ual reactivity of a mutant to a mAb was calculated according to the
formula: residual reactivity of a mutant to a mAb ? IC50of wild-type
PE38 to the mAb binding/IC50of the mutant to the mAb binding.
of the competition pattern shown in Fig. 2 (72). The mAbs with similar competition patterns are connected near the bottom of the tree and mAbs showing
dissimilar competition patterns go up in separate branches until they approach the top of the tree. The number of epitope groups was determined based on
the SI as described below. Values at each node represent percentage bootstrap support after 1000 replicates. The dotted line shows the cutoff value that
gives the 13 epitope subgroups as determined by the SI (B). B, The number of topographical epitopes was determined by the SI as described in Ref. 72.
SIs were defined according to the formula below and plotted against the number of epitopes:
Characterization of the topographical epitope groups identified in the mapping experiment. A, A dendrogram generated by cluster analysis
i ? 1
BSi? ?Hi? COg?
100 ? COg? g
where SI(g) is the SI for g groups of mAbs. Each epitope group (1, 2, . . . , g) joins to another group at each height in the dendrogram (H1,2, . . . , g).
COgis the highest cutoff value of the height that makes g ? 1 epitope groups of mAbs. BS1,2, . . . , gare the bootstrap percentages of the nodes where the
1,2, . . . , g epitope groups are made. The group number for the first peak (7 epitopes) was taken as the epitope group number (blue) and the value for the
second peak was taken as the epitope groups including subgroups (green, 13 subepitopes). C, Biacore sensorgrams for the serial injections of different mAbs.
SS1P immunotoxin was covalently coupled to the sensor chip CM5. Each mAb was serially injected over the chip surface at 10 ?l/min. The mAb numbers
and corresponding epitopes are shown.
8827The Journal of Immunology
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ELISA development to detect anti-PE38 mAbs
Passive adsorption onto plastic surfaces often alters protein con-
formation by destroying epitopes or revealing cryptic ones (66–
68). To avoid these problems, we designed an ICC-ELISA in
which Ag-Ab interactions take place in solution. Once formed, the
mAb-PE38 complex is captured by a target ligand tethered to the
plastic surface. The mAb is then detected with an HRP-labeled
second Ab (Fig. 1). A total of 60 mAbs to PE38 were characterized
using this ELISA.
Production of a large panel of anti-PE38 mAbs
Because PE38 differs greatly from mammalian proteins in se-
quence and in structure, we hypothesized that Abs produced by
humans or mice would recognize many of the same B cell epitopes
(51–54) also, see below. To obtain mAbs that react with the native
surface of the PE38, we immunized mice with various immuno-
toxin preparations and saved only those hybridomas that reacted
with native PE38. Reactivity for native PE38 was detected using
the ICC-ELISA—see Materials and Methods and Fig. 1 for de-
tails. To obtain a broad set of Abs, we used a variety of conditions
and schedules for immunization, as summarized in Table II. In
addition to the active immunotoxins, we also used inactive immu-
notoxin mutants for some of the immunizations. We used either
E553 or R276, which are important for different events in the cy-
totoxicity pathway (59, 60).
As shown in Table II, 10 fusions produced 57 mAbs that reacted
with the surface of PE38. Along with three previous mAbs (70),
we obtained a panel of 60 mAbs. The epitope locations of the three
mAbs that had been previously produced (“P” row in Table II)
were reidentified in the new mAb panel. Abs from mice with two
different H2 haplotypes (H2dfor BALB/c and H2afor A/J) reacted
with a similar variety of epitopes, suggesting that the same B cell
epitopes were recognized with distinct T cell support. Different
fusions yielded mAbs with different average affinity constants
from 0.4 to 65.2 ? 108M?1as determined using Biacore (each
value will be shown in Fig. 7). These values are within general
levels of affinities of other mAbs to protein Ags. The difference in
affinities and the difference in epitopes suggest that individual
mAbs are distinct.
Topographical epitope mapping and evaluation of the
relationships between the epitope locations
To classify the mAbs based on the topographical relationship of
their epitopes, we measured the mutual competition of all possible
pairs of the mAbs (72). The results are summarized in Fig. 2. The
competition between any two mAbs was almost always mutual;
very few (86 of 3600, 2.4%) showed ?50% competition in only
one direction. This indicates that the competition pattern is pri-
marily determined by the overlapping of epitopes and not by a
Evaluation of epitope overlap derived from the frequency of competition
indicator pairs that showed competition (?50%) out of all possible pairs
between the individual members of the epitope groups. Ep2a was excluded in
this figure because this group has only one mAb. B, Evaluation of the
clustering of the epitopes by a computer simulation. By computer, 60 virtual
epitopes were created by 60 random selections of exposed residues of PE38
and by expansion of each to 60 atoms around the residue. The competitive
pattern was generated by the set of virtual epitopes and analyzed by a cluster
analysis as was done with the experimental data. The inset matrix shows a
clustering of 60 virtual epitopes. The same simulation was conducted 10 times
and the clustering quality is shown as a receiver operating characteristic curve
in the graph. Each open circle with bars represents the average and SD of the
10 times simulations for a series of number of epitope groups. The experi-
mental data are shown in a curve with solid circles.
The distribution of the topographical epitopes on PE38. A,
treated with immunotoxins. Human serum samples of LMB-9 or SS1P
immunotoxin-treated patients were analyzed in the competition assay (Fig.
2) used for mapping topographical epitopes. Strengths of competition are
shown as percentages in each box, which are shaded in different colors as
in Fig. 2. The serum samples before and after treatment were obtained
during phase I clinical trials (10, 57). The development of neutralizing Abs
(measured by bioassays) prevented further administration. Immunotoxin
treatment induced human Abs against the same epitopes as those identified
by the mouse mAb panel.
Epitopes recognized by human Abs produced in patients
8828 B CELL EPITOPES OF PE38
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conformational change in PE38 induced by the binding of mAbs.
The competition pattern was analyzed by cluster analysis (72),
which gave a hierarchical tree representing the clustering of
epitopes (Fig. 3A). Objective criteria for clustering can be obtained
by the heights of nodes and from the bootstrap values (reproduc-
ibility of the connection). We used the stability index (SI) with
these two parameters to determine the number of epitope groups
(Fig. 3B) (72). We identified 7 major topographical epitope groups
that can be further divided into 13 subgroups (1a, 1b, etc., as
shown in Fig. 3A).
We characterized the quality of the Ab grouping of the Abs in
several ways. First, to examine whether the epitopes recognized by
different mAbs exhibiting no mutual competition were sufficiently
distinct to allow additive binding, five mAbs reacting with four
different epitopes (1b, 5, 6a, and 7) were evaluated in multiple
binding assays using Biacore (Fig. 3C). In the sensorgram, the
binding of one mAb to PE38 blocked binding of a second mAb to
the same epitope, but allowed the additional binding of three dif-
ferent mAbs to distinct epitopes. The signals from four mAbs to
different epitopes were additive and the total signal obtained from
the sequential injection of the four mAbs was the same as that from
a mixture of the four mAbs (data not shown). We conclude that the
competition assay represents the isolated location of each epitope
on the surface of PE38.
Next, we evaluated the separation between epitope groups by
the frequency of competition between the groups (Fig. 4A). For
example, subgroup 4a that consists of eight mAbs can be evaluated
for grouping quality by testing 8 ? 8 ? 64 combinations of com-
petition; all of these showed ?50% competition. This indicates an
excellent overlap of the individual epitopes in this group. The
overlap of Ep4a (eight members) with Ep4b (three members) can
be evaluated by testing 8 ? 3 ? 2 ? 48 combinations of compe-
tition, and 19 (40%) are positive for the competition. This indicates
a modest overlap between these subgroups. Epitope 2a was ex-
cluded from this analysis because this group has only one mAb.
Overlaps of (?20%) are observed between only 7 pairs of epitope
subgroups of 132 (5.3%) (1a/1b, 1a/3a, 3a/3b, 4a/4b, 5/6b, 6a/6b,
and 6b/7 pairs). Therefore, the epitope subgroups are clearly dis-
crete with relatively little overlap, suggesting a limited number of
B cell epitopes on the PE38 molecule. To examine whether the
epitopes of the mAb panel are distributed randomly or whether
they are located in clusters, we generated artificial epitopes ran-
domly on the surface of PE38 and performed a virtual competition
assay. The selection of epitopes was conducted with exposed res-
idues on PE38 and the data were compared with the experimental
epitope mapping data. The inset in Fig. 4B shows a representative
competition pattern with 60 randomly created epitopes around ex-
posed residues on the surface of PE38. Superficially, the clustering
pattern did not look as discrete as the experimental data (Fig. 2).
However, for an objective evaluation of the quality of the cluster-
ing (epitope independence), ROC plots were introduced in which
the true positive rates (rates of competitions inside clusters) and
false positive rates (rates of competitions outside clusters) in dif-
ferent epitope group numbers (cluster number) are shown (Fig.
4B). Each clustering gives a point on the ROC curve. Discrete
locations of epitopes should produce higher true positive rates and
lower false positive rates in the competitive patterns under a series
of numbers of clusters. The results should produce a ROC curve
that lies close to the left-top corner of the graph. As shown in Fig.
4B, the ROC curve from the experimental data is far closer to the
left-top corner than the average of the ROC curves randomly gen-
erated in 10 simulations. This result indicates that the clustering
pattern generated from the experimental competition data has a
nonrandom distribution on the surface of PE38. Thus, the epitope
locations are clustered.
Characterization of epitopes recognized by human
To determine whether immunotoxin treatment induces Ab re-
sponses to the same epitopes as those identified by the mAbs,
anti-PE38 Abs from the sera of eight patients were analyzed by the
competition assay used in Fig. 2. Sera from patients with pancre-
atic, colon cancer, or mesothelioma, who had been treated with
immunotoxins LMB-9 or SS1P, were evaluated (10, 57). In these
clinical trials, Ab production is routinely monitored using bioas-
says that measure immunotoxin neutralization activity. Most pa-
tients with solid tumors produced neutralizing Abs after one cycle
of immunotoxin treatment. Fig. 5 shows a competition analysis of
paired serum samples. Before treatment, the sera contained almost
no specific Ab to any of the PE38 epitopes, as expected from the
very low neutralizing activity of pretreatment sera. In contrast, the
sera obtained after immunotoxin treatment contained anti-PE38
of neutralization is the percentage of recovery of cellular protein synthesis compared with immunotoxin-treated cells in the absence of mAb. Bars represent
SDs of four replicate cultures. The association of neutralizing activity to Ep1a is significant (p ? 0.001) by nonparametric statistics (Mann-Whitney U test).
Neutralizing activity of anti-PE38 mAbs. Abolishment of immunotoxin-dependent cell killing by incubation with each mAb. The percentage
8829 The Journal of Immunology
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Abs to every topographical epitope as shown by their ability to
inhibit the binding of the corresponding mAbs to each epitope.
Some differences in competition values within the positive range
were recorded depending on the patients and the epitopes. These
results show that immunotoxin treatment induces human Abs
against the epitopes identified by the mouse mAb panel.
Characteristics of anti-PE38 mAbs
The association of the topographical epitopes with toxin neutral-
ization activity and with the binding kinetics of individual mAbs
was also examined. Fig. 6 shows the neutralization of an immu-
notoxin directed to CD25 (M1dsFv-PE38) by incubating it with an
excess amount of each mAb. The sensitivity of this neutralization
assay is similar to those used in our clinical trials (8, 9, 12). Only
6 of 60 mAbs neutralized the immunotoxin cytotoxic activity by
?50% (nos. 43, 62, 57, 34, 83, and 7); Ab concentration was kept
sufficiently high so that failure of a mAb to neutralize the immu-
notoxin was not the result of poor binding due to the lower affinity.
Of the six mAbs, three (nos. 43, 62, and 57) are assigned to the
same epitope subgroup, Ep1a, and all mAbs assigned to Ep1a
showed neutralization activity.
The binding kinetics of each mAb were measured by capturing
mAbs through an anti-Fc secondary Ab that had been covalently
attached on a Biacore chip and by flowing each immunotoxin over
the mAbs in solution to avoid possible alterations of the paratopes
and epitopes. As shown in Fig. 7, the mAbs show ranges of asso-
ciation rate constants (1.1–160 ? 104M?1s?1), dissociation rate
constants (0.1–218 ? 10?4s?1), and affinity constants (0.1–174 ?
108M?1) except one mAb (no. 57) showing an extremely low
dissociation rate constant (0.003 ? 10?4s?1resulting in 5.3 ?
1011M?1for the affinity constant).
We statistically evaluated the results of neutralization assay and
binding kinetics data to determine whether any of these character-
istics are associated with specific topographical epitopes. We find
that Ep1a is significantly associated with the neutralizing activity
of the mAbs binding to this epitope. In addition, Ep1a is signifi-
cantly associated with slower dissociation rates of the mAbs result-
ing in high affinity. The neutralization activity is not explained by the
simple increase of the affinity because mAbs that react with different
analyzed using a Biacore biosensor under conditions that allowed both
paratopes and epitopes to be recognized in native PE38. The connection of
the association rate, dissociation rate and affinity constants with the topo-
graphical epitopes was analyzed by nonparametric statistics (Mann-Whit-
ney U test). 1 , Significantly higher; 2 , significantly lower (p ? 0.01). A
high dissociation rate constant of mAb 11 (218 ? 10?4s?1) is shown as
100 ? 10?4s?1in this figure for simplification.
Binding kinetics of anti-PE38 mAbs. Binding kinetics was
mutants of PE38. A, Examples of the competition assay using mAbs 35 and
42 and wild-type PE38 (BL22 immunotoxin). Fifty-percent inhibition con-
centrations (IC50) were calculated from each curve. B, A plot of the IC50as
a function of the binding affinity (KA) of each mAb used for the compe-
tition assay. The IC50of each mAb were in general agreement with the
affinity values (correlation coefficient between IC50and KA? ?0.75).
mAbs 35 and 42 are shown in this graph as a red closed circle and blue
closed circle, respectively. C and D, Examples of the determination of
residual reactivity in competition assays using mAbs 35 and 42. These two
mAbs with different affinities both belong to Ep1b group. The IC50values
of E285A, E327A, and wild-type PE38 were 32, 330, and 32 ng/ml for
mAb 35 (shown in C), and 510, 5100, and 500 ng/ml for mAb 42 (shown
in D), respectively. Despite the differences in the IC50values between the
two mAbs with different affinity, the ratios of the IC50(residual reactiv-
ity ? ratio of the affinity) to each mutant were close (1.00 for no. 35 and
0.98 for no. 42 to E285A; 0.10 for no. 35 and 0.10 for no. 42 to E327A)
because of the proximity of the epitopes. We used the residual reactivities
for pairs of mAbs and mutants to evaluate the location epitopes (reported
in Fig. 9).
Residual reactivity (the difference in affinity) of mAbs to
8830B CELL EPITOPES OF PE38
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epitopes with similar affinity do not neutralize immunotoxins effi-
ciently. Ep1b and Ep3b are recognized by mAbs with lower affinities
and those with higher affinities, respectively, although the changes in
association and dissociation rate constants are not significant. Ep2c
and Ep 5 and 7 are associated with slower and faster association rates
of the mAbs, respectively, although the differences are not large
enough to significantly change the affinities.
Localization of topographical epitopes using PE38 mutants
To locate each epitope on PE38, we produced a panel of 41 point
mutants of PE38 (see Materials and Methods) and tested their
reactivity with 40 representative mAbs that cover all 7 major
epitope groups and 12 of 13 subgroups. Each mutant immunotoxin
was tested for its reactivity with each mAb. We developed an assay
that quantified the interaction in solution where both PE38 and
mAbs should be in a native state. This assay gives a competition
curve with a pair of mAbs and each immunotoxin mutant (Fig.
8A). Under these conditions, the 50% inhibition concentration
(IC50) indicates the apparent Ab affinity (81). A good correlation
(correlation coefficient ? ?0.75) was observed between the com-
petition values and the affinities that had been determined in a
BIAcore analysis (Fig. 8B).
Fig. 8, C and D, shows two examples of competition assays
using two mAbs 35 and 42. These two mAbs both belong to the
Ep1b group but exhibit different binding affinities to PE38 (3.3 ?
108M?1KAfor no. 35 and 3.8 ? 107M?1KAfor no. 42). As
expected, wild-type PE38 showed different IC50s for these two
mAbs (32 ng/ml for no. 35 and 500 ng/ml for no. 42) (Fig. 8A). In
to the E285A mutant was comparable with its binding to wild-type
PE38 (IC50? 32 ng/ml for no. 35 and 510 ng/ml for no. 42), while
reactivity of the mAbs with the E327A mutant was ?10% of wild
type as shown by shifts of the competition curves to 10-fold higher
compared with wild type were similar (the ratios to E285A were 1.00
for no. 35 and 0.98 for no. 42; the ratios to E327A were 0.10 for no.
35 and 0.10 for no. 42). In this study, residual reactivity was deter-
a comprehensive assessment of loss of reactivity depending on the
location of epitopes. We determined the residual reactivities for all
pairs of mutants and mAbs.
Residual reactivity of each mAb with a series of PE38 mutants
The results of 40 mAbs assayed against 41 mutants are summa-
rized in Fig. 9 in a matrix format. Each mAb bound to most of the
PE38 mutants with the same affinity as to wild-type PE38 but
failed to bind to a few key mutants (62 of 1636, 3.8%).
are shown. Orange, blue, and gray cells indicate ?0.1 residual reactivity, ?0.1 residual reactivity, and not tested (NT), respectively. The mutants are aligned
by the location of the mutated residue from the N terminus (left) to the C terminus (right). The exposed areas of each mutated residue are shown in Å2
in the PE38 model. The next column to the mAb names shows the IC50of the wild type (BL22-immunotoxin) in this assay.
Residual reactivity of each mAb to individual mutants of PE38. Residual reactivities of each PE38 mutant (columns) with each mAb (rows)
8831 The Journal of Immunology
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Conversely, each mutant bound normally to most of the mAbs but
lost its reactivity with a few mAbs and those were usually assigned
to the same topographical epitopes. Overall, the mutations that
reduced binding are specific for each epitope group, indicating that
the structures of these mutants are altered in a restricted area rec-
ognized by the mAbs assigned to the same topographical epitope.
Importantly, this also confirms that the structure of the rest of the
molecule is not affected.
Table III lists the mutations that reduced the binding to mAbs in
an epitope-specific manner (?10% binding to more than two
mAbs assigned for the same epitope). We identified 14 PE38 mu-
tants whose mutated residues affected the binding of some mAbs
in this manner. The locations of 7 major epitope groups (9 of 12
subgroups tested) were identified by the 14 mutants. Two sub-
groups of Ep1 were identified by a single Q332A mutant, indicat-
ing the proximity of Ep1 subgroups. Ep3a and Ep3b were also
identified by another single mutant (R313A). With some muta-
tions, the loss of reactivity was detected for all the mAbs assigned
to the same topographical epitope (footnote c in Table III). It is
likely that the side chain of these residues form an important core
for each topographical epitope structure and their mutation com-
monly reduced the binding of all the mAbs assigned for the same
topographical epitopes. We identified such key residues for Ep1,
2c, 3, 5, and 7. None of the 14 mutations rendered an immunotoxin
inactive (Table III). These mutants exhibited toxicity for Ag-pos-
itive cells in the same range as the wild-type immunotoxin protein
although small gains and loses of cytotoxicity with some mutants
Location of epitopes on the PE38 structure
Because the loss of reactivity is related to the location of the to-
pographical epitopes on PE38, we were able to locate each epitope
group by mutations that affected mAb binding. Fig. 10 shows the
location of the 14 aa residues listed in Table III. As expected,
mutated residues identifying the same epitope are generally lo-
cated on a restricted area on the structure model. The epitopes
identified by the mutant panel (shown in color) were located at the
surface of both domains of PE38 (II and III). Mutants that were not
recognized by any mAb are shown in gray. The gray residues are
often located between epitopes, indicating that this mutant analysis
also showed that epitopes of mAbs make clusters and are not
evenly distributed on the PE38 surface.
To characterize the humoral response to PE38, we injected mice
with several PE38-based immunotoxins and derived hybridomas
from the responding B cells. The reactivities of 57 new and 3
existing mAbs were then mapped to the surface of PE38. Mutual
competition experiments indicate the presence of 7 major epitopes,
which can be further divided into 13 subgroups. These epitopes
were also recognized by human Abs induced in patients treated
with immunotoxins. Among the 13 subgroups, epitope E1a was
found to be the major neutralizing epitope. Also, when binding
affinities were measured, Abs to E1a included some of the highest
affinity mAbs that we isolated. Because our eventual goal is to
modify PE38 and render it less immunogenic, we were gratified to
learn that the B cell repertoire sees only 7 major epitopes and only
1 of these provokes strongly neutralizing Abs. Thus, the number of
epitope sites that will need to be modified seems “manageable.” In
part, this may be due to the existence of antigenic hot spots. Our
computer analysis and the mapping experiments using a series of
on PE38 structure. The mutated amino acids that
decreased the binding to mAbs in an epitope-spe-
cific manner (listed in Table III) are shown in dif-
ferent colors on a structural model of PE38. The
model was made by extracting residues present in
PE38 (251–364 and 381–605) from the crystal
structure of PE (62). The clustered residues in dif-
ferent colors indicate the location of each epitope.
Locations of the epitope residues
Table III. Epitope-related residues of PE38 identified by the
corresponding point mutants with reduced reactivities with
mAbs against each epitope
% Cytotoxic Activity
(IC50) of the Mutant
to Wild Type in
aPoint mutant that showed ?10% residual reactivities against more than two
mAbs of the same epitope group. When a mutation was shared by two epitope sub-
groups, the subgroups are shown together.
bIC50of wild-type IT (BL22) to Raji cells ? 0.21 ng/ml.
cMutation that inhibited all the tested mAbs to the corresponding epitope.
8832B CELL EPITOPES OF PE38
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point mutants confirmed that the location of each epitope was clus-
tered and not random.
The most notable success in deimmunizing therapeutic proteins
has been the humanization of mouse mAbs by various means in-
cluding mutagenesis and CDR grafting, where regions specific to
mouse Ig were replaced with their human counterparts (44, 82).
These successes have focused attention on the foreignness of pro-
teins as a major obstacle to achieving success in the clinic (3, 5).
Here, we present evidence for the presence of a limited number of
epitope clusters on the surface of a bacterial protein, PE38. Our
results suggest that a foreign protein may be made less immuno-
genic by replacing structures that are likely to be epitopes with
those that are unlikely to become epitopes, although we have not
identified the structural characteristics that determine the tendency
to become epitopes. In this study, we successfully produced PE38
point mutants that abolish the binding of anti-PE38 mAbs in an
epitope-specific manner. We also produced many mutants that
caused no change in the binding of any of the mAbs. A compre-
hensive comparison of the structural difference between the areas
altered by these mutations might help to disclose common struc-
tural features that determine the likelihood of a region becoming
an immunogenic site.
The 14 mutants reported here with diminished binding to mAbs
(Table III) are predicted to be less antigenic forms of PE38 in an
epitope basis. The mutants will be used in the development of
immunotoxins with reduced immunogenicity. Deimmunization of
functional proteins is only possible if mutant versions retain bio-
logical activity. We found that all the 14 individual PE38 mutants
retained substantial cytotoxic activity, despite the change of hy-
drophilic residues to either alanine or glycine. Ep1a that was
shown to be associated with strong neutralizing activity (Fig. 6)
was mapped to a region around Q332. It is particularly interesting
that mutant Q332A does not bind to any of Ep1a mAbs (all are
neutralizing) and yet retains full cytotoxicity. This residue is dis-
tant from the six functionally important regions previously iden-
tified: 276–279 (60), around W281 (83), 350–355 (84), around
Y470 (85), around E553 (59), and 609–613 (86). Another poten-
tially important mutation is R490A that abolished the binding of
Ep5 mAbs. We recently reported that this mutation increases spe-
cific cytotoxicity of some PE38-based immunotoxins (87). This
mutant could be useful both to diminish antigenicity and to in-
crease the efficacy of immunotoxins.
In this study, we characterized Abs to epitopes exposed on the
native surface of PE38, which should be useful for the prediction of
Ab formation in patients treated with immunotoxins. Our approach to
the selection of mutants and the quantitative assay that measures re-
sidual reactivity enabled us to scan the whole protein surface for B
cell epitopes with a relatively small number of mutants. The identi-
fication of topographical epitopes on the surface of protein Ags fol-
lowed by determining their location using point mutants could be the
basis for a strategy to deimmunize foreign proteins.
We thank Dr. Sookhee Bang for preparation of the R490A mutant of PE38,
and Anna Mazzuca and Dawn A. Walker for their editorial assistance.
I. Pastan and D. J. FitzGerald are partial owners of a non-U.S. patent on the
use of PE38 to produce immunotoxins. The patent is currently licensed to
Neopharm and IVAX for use in countries outside the United States.
1. Crommelin, D. J., G. Storm, R. Verrijk, L. de Leede, W. Jiskoot, and
W. E. Hennink. 2003. Shifting paradigms: biopharmaceuticals versus low mo-
lecular weight drugs. Int. J. Pharm. 266: 3–16.
2. Pavlou, A. K., and J. M. Reichert. 2004. Recombinant protein therapeutics-suc-
cess rates, market trends and values to 2010. Nat. Biotechnol. 22: 1513–1519.
3. Schellekens, H. 2002. Immunogenicity of therapeutic proteins: clinical implica-
tions and future prospects. Clin. Ther. 24: 1720–1740.
4. Bellanti, J. A., and R. E. Green. 1971. Immunological reactivity: expression of
efficiency in elimination of foreignness. Lancet 2: 526–529.
5. Rosenberg, A. S. 2003. Immunogenicity of biological therapeutics: a hierarchy of
concerns. Dev. Biol. 112: 15–21.
6. Chaudhary, V. K., C. Queen, R. P. Junghans, T. A. Waldmann, D. J. FitzGerald,
and I. Pastan. 1989. A recombinant immunotoxin consisting of two antibody
variable domains fused to Pseudomonas exotoxin. Nature 339: 394–397.
7. Brinkmann, U., J. Buchner, and I. Pastan. 1992. Independent domain folding of
Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain
connections. Proc. Natl. Acad. Sci. USA 89: 3075–3079.
8. Kreitman, R. J., W. H. Wilson, J. D. White, M. Stetler-Stevenson, E. S. Jaffe,
S. Giardina, T. A. Waldmann, and I. Pastan. 2000. Phase I trial of recombinant
immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malig-
nancies. J. Clin. Oncol. 18: 1622–1636.
9. Kreitman, R. J., W. H. Wilson, K. Bergeron, M. Raggio, M. Stetler-Stevenson,
D. J. FitzGerald, and I. Pastan. 2001. Efficacy of the anti-CD22 recombinant
immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N. Engl.
J. Med. 345: 241–247.
10. Hassan, R., M. R. Lerner, D. Benbrook, S. A. Lightfoot, D. J. Brackett,
Q. C. Wang, and I. Pastan. 2002. Antitumor activity of SS(dsFv)PE38 and
SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human
gynecologic cancers grown in organotypic culture in vitro. Clin. Cancer Res. 8:
11. FitzGerald, D. J., R. Kreitman, W. Wilson, D. Squires, and I. Pastan. 2004.
Recombinant immunotoxins for treating cancer. Int. J. Med. Microbiol. 293:
12. Kreitman, R. J., D. R. Squires, M. Stetler-Stevenson, P. Noel, D. J. FitzGerald,
W. H. Wilson, and I. Pastan. 2005. Phase I trial of recombinant immunotoxin
RFB4(dsFv)-PE38 (BL22) in patients with B-cell malignancies. J. Clin. Oncol.
13. Pastan, I., R. Hassan, D. J. FitzGerald, and R. J. Kreitman. 2006. Immunotoxin
therapy of cancer. Nat. Rev. Cancer 6: 559–565.
14. Roscoe, D. M., L. H. Pai, and I. Pastan. 1997. Identification of epitopes on a mutant
form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas
exotoxin containing immunotoxins. Eur. J. Immunol. 27: 1459–1468.
15. Hassan, R., J. Williams-Gould, T. Watson, L. Pai-Scherf, and I. Pastan. 2004.
Pretreatment with rituximab does not inhibit the human immune response against
the immunogenic protein LMB-1. Clin. Cancer Res. 10: 16–18.
16. Molineux, G. 2003. Pegylation: engineering improved biopharmaceuticals for
oncology. Pharmacotherapy 23: 3S–8S.
17. Veronese, F. M., and G. Pasut. 2005. PEGylation, successful approach to drug
delivery. Drug Discov. Today 10: 1451–1458.
18. Benhar, I., Q. C. Wang, D. FitzGerald, and I. Pastan. 1994. Pseudomonas exo-
toxin A mutants: replacement of surface-exposed residues in domain III with
cysteine residues that can be modified with polyethylene glycol in a site-specific
manner. J. Biol. Chem. 269: 13398–13404.
19. De Groot, A. S., P. M. Knopp, and W. Martin. 2005. De-immunization of ther-
apeutic proteins by T-cell epitope modification. Dev. Biol. 122: 171–194.
20. Tangri, S., B. R. Mothe, J. Eisenbraun, J. Sidney, S. Southwood, K. Briggs,
J. Zinckgraf, P. Bilsel, M. Newman, R. Chesnut, et al. 2005. Rationally engi-
neered therapeutic proteins with reduced immunogenicity. J. Immunol. 174:
21. Laroche, Y., S. Heymans, S. Capaert, F. De Cock, E. Demarsin, and D. Collen.
2000. Recombinant staphylokinase variants with reduced antigenicity due to
elimination of B-lymphocyte epitopes. Blood 96: 1425–1432.
22. Bresson, D., M. Pugniere, F. Roquet, S. A. Rebuffat, B. Guyen, M. Cerutti,
J. Guo, S. M. McLachlan, B. Rapoport, V. Estienne, et al. 2004. Directed mu-
tagenesis in region 713–720 of human thyroperoxidase assigns 713KFPED717
residues as being involved in the B domain of the discontinuous immunodomi-
nant region recognized by human autoantibodies. J. Biol. Chem. 279:
23. Mayer, A., S. K. Sharma, B. Tolner, N. P. Minton, D. Purdy, P. Amlot,
G. Tharakan, R. H. Begent, and K. A. Chester. 2004. Modifying an immunogenic
epitope on a therapeutic protein: a step towards an improved system for antibody-
directed enzyme prodrug therapy (ADEPT). Br. J. Cancer 90: 2402–2410.
24. Pieters, J. 2000. MHC class II-restricted antigen processing and presentation.
Adv. Immunol. 75: 159–208.
25. Marsh, S. G. E., P. Parham, and L. D. Barber, eds. 2000. The HLA Facts Book.
Academic Press, London.
26. Brons, N. H., A. Blaich, K. H. Wiesmuller, F. Schneider, G. Jung, and
C. P. Muller. 1996. Hierarchic T-cell help to non-linked B-cell epitopes. Scand.
J. Immunol. 44: 478–484.
27. Rosenberg, J. S., and M. Z. Atassi. 1997. Intersite helper function of T cells
specific for a protein epitope that is not recognized by antibodies. Immunol.
Invest. 26: 473–489.
28. Robak, T. 2001. Cladribine in the treatment of chronic lymphocytic leukemia.
Leuk. Lymphoma 40: 551–564.
29. White, T. J., I. M. Ibrahimi, and A. C. Wilson. 1978. Evolutionary substitutions
and the antigenic structure of globular proteins. Nature 274: 92–94.
30. Berzofsky, J. A. 1985. Intrinsic and extrinsic factors in protein antigenic struc-
ture. Science 229: 932–940.
8833The Journal of Immunology
by guest on June 13, 2013
31. Geysen, H. M., J. A. Tainer, S. J. Rodda, T. J. Mason, H. Alexander, Download full-text
E. D. Getzoff, and R. A. Lerner. 1987. Chemistry of antibody binding to a protein.
Science 235: 1184–1190.
32. Benjamin, D. C. 1995. B-cell epitopes: fact and fiction. Adv. Exp. Med. Biol. 386:
33. Davies, D. R., and G. H. Cohen. 1996. Interactions of protein antigens with
antibodies. Proc. Natl. Acad. Sci. USA 93: 7–12.
34. Sathiamurthy, M., B. Peters, H. H. Bui, J. Sidney, J. Mokili, S. S. Wilson,
W. Fleri, D. L. McGuinness, P. E. Bourne, and A. Sette. 2005. An ontology for
immune epitopes: application to the design of a broad scope database of immune
reactivities. Immunome Res. 1: 2.
35. Calef, C., C. Kuiken, J. Szinger, B. Gaschen, W. Abfalterer, M. Zhanf, N. Tao,
R. Funkhouser, K. Yusim, M. Flynn, et al. 2005. Gateway to tools of HIV and
HCV databases. In HIV Mol. Immunol. Los Alamos National Laboratory, The-
oretical Biology and Biophysics, Los Alamos, pp. 33–56.
36. van Regenmortel, M. H. V. 1992. Molecular dissection of protein antigens. In
Structure of Antigens, Vol. I. M. H. V. van Regenmortel, ed. CRC Press, Boca
Raton, FL, pp. 1–27.
37. Padlan, E. A. 1992. Structure of protein epitopes deduced from x-ray crystallog-
raphy. In Structure of Antigens, Vol. I. M. H. V. van Regenmortel, ed. CRC Press,
Boca Raton, FL, pp. 29–42.
38. Braden, B. C., and R. J. Poljak. 1995. Structural features of the reactions between
antibodies and protein antigens. FASEB J. 9: 9–16.
39. Collis, A. V. J., A. P. Brouwer, and A. C. R. Martin. 2003. Analysis of the antigen
combining site: correlations between length and sequence composition of the
hypervariable loops and the nature of the antigen. J. Mol. Biol. 325: 337–354.
40. Livesay, D. R., and S. Subramaniam. 2004. Conserved sequence and structure
association motifs in antibody-protein and antibody-hapten complexes. Protein
Eng. Des. Sel. 17: 463–472.
41. Carter, J. M. 1994. Epitope prediction methods. Methods Mol. Biol. 36: 193–206.
42. Alix, A. J. 1999. Predictive estimation of protein linear epitopes by using the
program PEOPLE. Vaccine 18: 311–314.
43. Kulkarni-Kale, U., S. Bhosle, and A. S. Kolaskar. 2005. CEP: a conformational
epitope prediction server. Nucleic Acids Res. 33: W168–W171.
44. Hwang, W. Y., and J. Foote. 2005. Immunogenicity of engineered antibodies.
Methods 36: 3–10.
45. Roscoe, D. M., S. H. Jung, I. Benhar, L. Pai, B. K. Lee, and I. Pastan. 1994.
Primate antibody response to immunotoxin: serological and computer-aided anal-
ysis of epitopes on a truncated form of Pseudomonas exotoxin. Infect. Immun. 62:
46. Barlow, D. J., M. S. Edwards, and J. M. Thornton. 1986. Continuous and dis-
continuous protein antigenic determinants. Nature 322: 747–748.
47. Ito, H. O., T. Nakashima, T. So, M. Hirata, and M. Inoue. 2003. Immunodomi-
nance of conformation-dependent B-cell epitopes of protein antigens. Biochem.
Biophys. Res. Commun. 308: 770–776.
48. Laver, W. G., G. M. Air, R. G. Webster, and S. J. Smithgill. 1990. Epitopes on
protein antigens—misconceptions and realities. Cell 61: 553–556.
49. Cunningham, B. C., and J. A. Wells. 1989. High-resolution epitope mapping of
hGH-receptor interactions by alanine-scanning mutagenesis. Science 244:
50. Wells, J. A. 1991. Systematic mutational analyses of protein-protein interfaces.
Methods Enzymol. 202: 390–411.
51. Atassi, M. Z., B. Z. Dolimbek, M. Hayakari, J. L. Middlebrook, B. Whitney, and
M. Oshima. 1996. Mapping of the antibody-binding regions on botulinum neu-
rotoxin H-chain domain 855–1296 with antitoxin antibodies from three host spe-
cies. J. Protein Chem. 15: 691–700.
52. Goding, J. W. 1996. Nature of antigens. In Monoclonal Antibodies: Principles
and Practice. Academic Press, San Diego, pp. 50–71.
53. Bugelski, P. J., and G. Treacy. 2004. Predictive power of preclinical studies in
animals for the immunogenicity of recombinant therapeutic proteins in humans.
Curr. Opin. Mol. Ther. 6: 10–16.
54. Atassi, M. Z., and B. Z. Dolimbek. 2004. Mapping of the antibody-binding re-
gions on the HN-domain (residues 449–859) of botulinum neurotoxin A with
antitoxin antibodies from four host species: full profile of the continuous anti-
genic regions of the H-chain of botulinum neurotoxin A. Protein J. 23: 39–52.
55. Kreitman, R. J., P. Bailon, V. K. Chaudhary, D. J. FitzGerald, and I. Pastan. 1994.
Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudo-
monas exotoxin produce complete regression in mice of an interleukin-2 recep-
tor-expressing human carcinoma. Blood 83: 426–434.
56. Onda, M., R. J. Kreitman, G. Vasmatzis, B. Lee, and I. Pastan. 1999. Reduction
of the nonspecific animal toxicity of anti-Tac(Fv)-PE38 by mutations in the
framework regions of the Fv which lower the isoelectric point. J. Immunol. 163:
57. Hausner, P. F., J. E. Karp, N. L. Edelma, N. Kennedy, M. Tathineni, M. Willingham,
L. H. Pai-Scherf, R. J. Kreitman, and I. Pastan. 2003. Phase I study of LMB-9
(B3(dsFv)PE38), a recombinant disulfide stabilized anti-Lewis Y immunotoxin ad-
ministered by continuous infusion. Proc. Am. Soc. Clin. Oncol. 22: 184.
58. Nagata, S., M. Onda, Y. Numata, K. Santora, R. Beers, R. J. Kreitman, and
I. Pastan. 2002. Novel anti-CD30 recombinant immunotoxins containing disul-
fide-stabilized Fv fragments. Clin. Cancer Res. 8: 2345–2355.
59. Douglas, C. M., and R. J. Collier. 1987. Exotoxin A of Pseudomonas aeruginosa:
substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity
and enzymatic activity. J. Bacteriol. 169: 4967–4971.
60. Ogata, M., C. M. Fryling, I. Pastan, and D. J. FitzGerald. 1992. Cell-mediated
cleavage of Pseudomonas exotoxin between Arg279and Gly280generates the
enzymatically active fragment which translocates to the cytosol. J. Biol. Chem.
61. Li, M., F. Dyda, I. Benhar, I. Pastan, and D. R. Davies. 1995. The crystal structure
of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP:
conformational differences with the intact exotoxin. Proc. Natl. Acad. Sci. USA
62. Wedekind, J. E., C. B. Trame, M. Dorywalska, P. Koehl, T. M. Raschke,
M. McKee, D. FitzGerald, R. J. Collier, and D. B. McKay. 2001. Refined crys-
tallographic structure of Pseudomonas aeruginosa exotoxin A and its implica-
tions for the molecular mechanism of toxicity. J. Mol. Biol. 314: 823–837.
63. Pastan, I., R. Beers, and T. K. Bera. 2004. Recombinant immunotoxins in the
treatment of cancer. Methods Mol. Biol. 248: 503–518.
64. Salvatore, G., S. Nagata, M. Billaud, M. Santoro, G. Vecchio, and I. Pastan. 2002.
Generation and characterization of novel monoclonal antibodies to the Ret re-
ceptor tyrosine kinase. Biochem. Biophys. Res. Commun. 294: 813–817.
65. Ise, T., H. Maeda, K. Santora, L. Xiang, R. J. Kreitman, I. Pastan, and S. Nagata.
2005. Immunoglobulin superfamily receptor translocation associated 2 protein on
lymphoma cell lines and hairy cell leukemia cells detected by novel monoclonal
antibodies. Clin. Cancer Res. 11: 87–96.
66. Mierendorf, R. C., Jr., and R. L. Dimond. 1983. Functional heterogeneity of
monoclonal antibodies obtained using different screening assays. Anal. Biochem.
67. Schwab, C., and H. R. Bosshard. 1992. Caveats for the use of surface-adsorbed
protein antigen to test the specificity of antibodies. J. Immunol. Methods 147:
68. Mire-Sluis, A. R., Y. C. Barrett, V. Devanarayan, E. Koren, H. Liu, M. Maia,
T. Parish, G. Scott, G. Shankar, E. Shores, et al. 2004. Recommendations for the
design and optimization of immunoassays used in the detection of host antibodies
against biotechnology products. J. Immunol. Methods 289: 1–16.
69. Nagata, S., G. Salvatore, and I. Pastan. 2003. DNA immunization followed by a
single boost with cells: a protein-free immunization protocol for production of
monoclonal antibodies against the native form of membrane proteins. J. Immunol.
Methods 280: 59–72.
70. Ogata, M., I. Pastan, and D. FitzGerald. 1991. Analysis of Pseudomonas exotoxin
activation and conformational changes by using monoclonal antibodies as probes.
Infect. Immun. 59: 407–414.
71. Nagata, S., K. Yamamoto, Y. Ueno, T. Kurata, and J. Chiba. 1991. Preferential
generation of monoclonal IgG-producing hybridomas by use of vesicular stoma-
titis virus-mediated cell fusion. Hybridoma 10: 369–378.
72. Nagata, S., Y. Numata, M. Onda, T. Ise, Y. Hahn, B. Lee, and I. Pastan. 2004.
Rapid grouping of monoclonal antibodies based on their topographical epitopes
by a label-free competitive immunoassay. J. Immunol. Methods 292: 141–155.
73. Canziani, G. A., S. Klakamp, and D. G. Myszka. 2004. Kinetic screening of
antibodies from crude hybridoma samples using Biacore. Anal. Biochem. 325:
74. Laricchia, R. L., P. Uboldi, S. Marcovina, R. P. Revoltella, and A. L. Catapano.
2001. Epitope mapping analysis of apolipoprotein B-100 using a surface plasmon
resonance-based biosensor. Biosens. Bioelectron. 16: 963–969.
75. Lee, B., and F. M. Richards. 1971. The interpretation of protein structures: es-
timation of static accessibility. J. Mol. Biol. 55: 379–400.
76. Beam, C. A. 1998. Analysis of clustered data in receiver operating characteristic
studies. Stat. Methods Med. Res. 7: 324–336.
77. Almagro, J. C. 2004. Identification of differences in the specificity-determining
residues of antibodies that recognize antigens of different size: implications for
the rational design of antibody repertoires. J. Mol. Recognit. 17: 132–143.
78. Kasturi, S., A. Kihara, D. FitzGerald, and I. Pastan. 1992. Alanine scanning
mutagenesis identifies surface amino acids on domain II of Pseudomonas exo-
toxin required for cytotoxicity, proper folding, and secretion into periplasm.
J. Biol. Chem. 267: 23427–23433.
79. Berzofsky, J. A., and A. N. Schechter. 1981. The concepts of crossreactivity and
specificity in immunology. Mol. Immunol. 18: 751–763.
80. Miller, J. J., and R. Valdes. 1992. Methods for calculating cross-reactivity in
immunoassays. J. Clin. Immunoassay 15: 97–107.
81. Friguet, B., A. F. Chaffotte, L. Djavadi-Ohaniance, and M. E. Goldberg. 1985. Mea-
surements of the true affinity constant in solution of antigen-antibody complexes by
enzyme-linked immunosorbent assay. J. Immunol. Methods 77: 305–319.
82. Kashmiri, S. V., R. De Pascalis, N. R. Gonzales, and J. Schlom. 2005. SDR
grafting—a new approach to antibody humanization. Methods 36: 25–34.
83. Zdanovsky, A. G., M. Chiron, I. Pastan, and D. J. FitzGerald. 1993. Mechanism
of action of Pseudomonas exotoxin: identification of a rate-limiting step. J. Biol.
Chem. 268: 21791–21799.
84. Siegall, C. B., M. Ogata, I. Pastan, and D. J. FitzGerald. 1991. Analysis of
sequences in domain II of Pseudomonas exotoxin A which mediate translocation.
Biochemistry 30: 7154–7159.
85. Yates, S. P., and A. R. Merrill. 2004. Elucidation of eukaryotic elongation fac-
tor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exo-
toxin A. Biochem. J. 379: 563–572.
86. Chaudhary, V. K., Y. Jinno, D. FitzGerald, and I. Pastan. 1990. Pseudomonas
exotoxin contains a specific sequence at the carboxyl terminus that is required for
cytotoxicity. Proc. Natl. Acad. Sci. USA 87: 308–312.
87. Bang, S., S. Nagata, M. Onda, R. J. Kreitman, and I. Pastan. 2005. HA22
(R490A) is a recombinant immunotoxin with increased antitumor activity without
an increase in animal toxicity. Clin. Cancer Res. 11: 1545–1550.
8834 B CELL EPITOPES OF PE38
by guest on June 13, 2013