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of June 18, 2017.
Molecular Characterization of the
Anti-Idiotypic Immune Response of a
Relapse-Free Neuroblastoma Patient
following Antibody Therapy: A Possible
Vaccine against Tumors of Neuroectodermal
Origin?
Martina M. Uttenreuther-Fischer, Jörg A. Krüger and Peter
Fischer
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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 © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 176:7775-7786; ;
doi: 10.4049/jimmunol.176.12.7775
http://www.jimmunol.org/content/176/12/7775
The Journal of Immunology
Molecular Characterization of the Anti-Idiotypic Immune
Response of a Relapse-Free Neuroblastoma Patient following
Antibody Therapy: A Possible Vaccine against Tumors of
Neuroectodermal Origin?1
Martina M. Uttenreuther-Fischer,2* Jörg A. Krüger,2* and Peter Fischer3*†
T
he outcome of the advanced or relapsed stage IV neuroblastoma is still dismal regardless of continuing therapeutic efforts. Drawbacks of an intensification of chemotherapy include secondary malignancies or therapy-related deaths due
to the increased toxicity of such a regimen (1–3). Passive immunotherapy with murine or human/mouse chimeric Abs directed
against GD2, a disialoganglioside overexpressed on neuroblastoma
cells, in phase I/II trials demonstrated complete remissions and prolonged event-free survival in some neuroblastoma patients (4 –15).
Initially, the clinical efficacy of native anti-GD2 Ab therapies was
mainly attributed to their capability of causing tumor cell killing by
Ab-dependent, cell-mediated cytotoxicity and complement-dependent
cytotoxicity (16). However, although the infused Abs were cleared
from circulation after six half-lives, which is ⬃17 days in children for
mAb ch14.18, clinical remissions were of longer duration, implying
that additional antitumor defense mechanisms must have been triggered (17, 18). The potential role of a “vaccination-like” effect in
long-term neuroblastoma survivors suggested by Cheung et al. and
other research teams (4, 10, 19 –21) indicated that patients with an
*Charité, Hochschulmedizin Berlin, Campus Virchow Klinikum, Children’s Hospital,
Dept. General Pediatrics, Berlin, Germany; and †University of Applied Sciences,
Fachbereich V Life Sciences and Technology, Biotechnology, Berlin, Germany
Received for publication December 30, 2005. Accepted for publication March
31, 2006.
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 research was supported in part by Deutsche Forschungsgemeinschaft Grants
GA 167/6-1 and 6-2 (to M.M.U.-F. and P. F.) and Charité Grant 2001-749 (to J.A.K.).
2
M.M.U.-F. and J.A.K. contributed equally to this work and should be considered
first authors.
3
Address correspondence and reprint requests to Dr. Peter Fischer, University of
Applied Sciences, Fachbereich V Life Sciences and Technology, Biotechnology,
Seestrasse 64, 13347 Berlin, Germany. E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
immune response benefited from passive immunotherapy. Similar
results were confirmed for the adult patient population (22–24).
Human anti-mouse Abs (HAMAs)4 seemed to preclude repetitive
therapeutic use of murine Abs by a diminished tumor targeting,
accelerated clearance, and reduction of direct anti-tumor effects (9, 20,
25–27). Some of these problems were addressed by decreasing the
size and the xenogeneic protein parts of Abs (28, 29). However, even
with chimerized or humanized Abs, immunogenicity was still observed, although it neither limited treatment nor made patients prone
to increased toxicity (14, 30 –32).
In an analysis of a larger patient population of neuroblastoma
survivors, Cheung et al. (6) proposed the hypothesis that low, transient levels of HAMAs, which are mainly directed against the constant regions of murine Abs, were positively correlated with patient survival. Immunogenicity of diagnostic or therapeutic mAbs
was not disadvantageous for the patient but appeared beneficial by
triggering an activation of the idiotypic network, as proposed by
Jerne in 1974 (33). Accordingly, Ag-specific idiotypic (therapeutic) Ab (Ab1) induces production of the anti-idiotypic antibody
(Ab2) by unique antigenic determinants of its Ag combining site.
Anti-idiotypic Ab2s include three groups of Abs: 1) Ab2␣,
which binds to the variable region of Ab1 but does not fit into the
paratopes of Ab1 or inhibit Ag-binding; 2) Ab2␤, which forms a
mirror or internal image of the three-dimensional structure and the
interactions of the Ag recognized by Ab1 (34); and 3) Ab2␥, which
inhibits Ag binding but does not mirror the Ag. Ab2␤ itself can
induce an immune response termed Ab3, the so-called anti-antiidiotypic Ab, which also binds the original tumor-associated Ag.
Therefore, Ab2␤ can be used as a surrogate Ag, e.g., as a vaccine.
4
Abbreviations used in this paper: HAMA, human anti-mouse Abs; Ab1, idiotype
(therapeutic) Ab; Ab2, anti-idiotypic Ab; Ab3, anti-anti-idiotypic Ab; BMT, bone
marrow transplant; IVIG, pooled human i.v. Ig; R/S, ratio of replacement to silent
mutations; scFv, single-chain variable fragment.
0022-1767/06/$02.00
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Neuroblastoma treatment with chimeric antidisialoganglioside GD2 Ab ch14.18 showed objective antitumor responses. Production
of anti-idiotypic Abs (Ab2) against ch14.18 (Ab1) in some cases was positively correlated with a more favorable prognosis.
According to Jerne’s network theory, a subset of anti-idiotypic Abs (Ab2␤) carries an “internal image” of the Ag and induces Abs
(Ab3) against the original Ag. The molecular origin of an anti-idiotypic Ab response in tumor patients was not investigated
previously. To clone anti-idiotypic Abs, B cells of a ch14.18-treated neuroblastoma patient with Ab2 serum reactivity were used
to construct Ab phage display libraries. After repeated biopannings on ch14.18 and its murine relative, anti-GD2 mAb 14G2a, we
selected 40 highly specific clones. Sequence analysis revealed at least 10 of 40 clones with different Ig genes. Identities to putative
germline genes ranged between 94.90 and 100% for VH and between 93.90 and 99.60% for VL. An overall high rate of replacement
mutations suggested a strong Ag-driven maturation of the anti-idiotypic Abs. Two clones that were analyzed further, GK2 and
GK8, inhibited binding of ch14.18 to GD2 just as the patient’s serum did. GK8 alone inhibited >80% of the patient’s anti-idiotypic
serum Abs in binding to ch14.18. Rabbits vaccinated with GK8 or GK2 (weaker) produced Ab3 against the original target Ag
GD2. GK8 may be useful as a tumor vaccine for CD3-positive tumors. The Journal of Immunology, 2006, 176: 7775–7786.
7776
Materials and Methods
Patient selection
Sera for anti-idiotypic screening were obtained from nine high risk neuroblastoma patients treated with ch14.18 according to the NB97 protocol of
the Society for Pediatric Oncology and Hematology (German abbreviation
GPOH) 具www.kinderkrebsinfo.de/e1662/e7722/e5408/index_ger.html典 at
the Charité Children’s Hospital Berlin, Germany (28, 54). Serum sampling
was performed after informed consent was obtained from all patients or
their guardians, respectively, according to the Helsinki Declaration.
Initial screening of patients’ sera was done on 14G2a, a murine antiGD2 Ab sharing the same variable region with ch14.18, but it was never
given to our patients therapeutically (55). Patients’ sera were also tested on
OKT3, a murine anti-CD3 mAb and a member of the IgG2a subclass like
14G2a (S. Gillies, unpublished observation) (56). Because the amino acid
sequence of the framework region of OKT3 and 14G2a are identical, nonspecific binding of HAMAs and the anti-framework Abs that only react
with epitopes of 14G2a outside the CDRs could be largely excluded.
Screening assay for Ab2
Microtiter plates (Costar; Corning) were coated with 14G2a (250 ng/well),
a murine anti-GD2 Ab (BioTechnetics) in PBS at 4°C overnight. After
washing with PBS containing 0.2% casein, plates were blocked with PBS
containing 1% casein for 1 h at 37°C. Serial dilutions of patient sera in PBS
were added and incubated for 1 h at 37°C. After washing with PBS, bound
anti-idiotypic and potential anti-mouse Ig Abs were detected by a peroxidase-labeled, goat anti-human Fc fragment (Jackson ImmunoResearch
Laboratories). ELISAs were performed twice, and samples were tested in
duplicate or triplicate.
GD2 binding inhibition by Ab2 in patients’ sera
Microtiter plates were coated with GD2 (Pierce) dissolved in methanol at
a concentration of 150 ng/well for 2 h until the methanol had evaporated.
Plates were blocked and washed as described above. Patient samples were
added at serial dilutions together with biotinylated ch14.18 (250 ng/well)
and incubated at 37°C for 1 h. Ch14.18, a chimeric anti-GD2-mAb (BioInvent) was exclusively supplied for i.v. mAb therapy. Leftover i.v. supplies were used for in vitro studies. For biotinylation, a commercial kit
(Sigma-Aldrich) was used following the manufacturer’s instructions. After
washing with PBS, bound ch14.18 was detected with peroxidase-labeled
streptavidin (Jackson ImmunoResearch Laboratories).
Determination of Ig isotypes and subclasses of anti-idiotypic
antibodies
Microtiter plates were coated overnight at 4°C with 700 ng of 14G2a in
PBS per well. Plates were blocked and washed as described above. Patient
serum (patient 1) containing anti-idiotypic Abs was added at a dilution of
1/250 for 1 h at 37°C. After washing with PBS, biotinylated Abs against
the different Ig isotypes and IgG subclasses (BD Pharmingen) as well as
against ␬ and ␭ L chains (DakoCytomation) were applied according to the
manufacturer’s recommendations. Pooled human i.v. IgG (IVIG) (Sandoglobulin; Chiron-Behring) was used as a control.
GD2 binding inhibition by purified Fab
Microtiter plates were coated with GD2, blocked, and washed as described
above. Purified Fabs were added at concentrations between 0 and 1.5 ␮g
together with biotinylated ch14.18 (250 ng/well) and incubated at 37°C for 1 h.
After washing with PBS, bound ch14.18 was detected with peroxidase-labeled
streptavidin. Purification of Fab is described in detail below in this section.
Competition assay of purified Fab and patient serum (Ab2␤) on
14G2a
Microtiter plates were coated with 14G2a as described above. Purified Fabs
were added at concentrations between 0 and 6 ␮g/well together with the
serum of patient 1 at a dilution of 1/20,000 to adjust for IgG concentration
and incubated at 37°C for 1 h. After washing with PBS, bound antiidiotypes and potential anti-mouse Ig Abs of the patient were detected
using a peroxidase-labeled goat anti-human Fc Ab (Jackson ImmunoResearch Laboratories).
Binding of Ab3 from rabbit serum to GD2
Microtiter plates were coated with GD2. The IgG content of the different
rabbit sera was determined by a sandwich ELISA using a coating of antirabbit IgG/IgM/IgA (BioTeZ) and a secondary anti-rabbit IgG HRP Ab
(DakoCytomation). A serial dilution of a rabbit-anti-goat Ab served as a
standard (Jackson ImmunoResearch Laboratories). For calculations, the
program Revelation (Bio-Rad) was used. A rabbit serum volume containing ⬃35 ␮g of rabbit IgG was added to each well. Sera from the three
rabbits immunized with Fab phage were preincubated for 2 h at 37°C on a
microtiter plate precoated with pooled human Abs (1 ␮g/well). Thereafter,
supernatants were transferred to GD2-coated microtiter plates, incubated
for 1 h at 37°C, and washed with PBS. Bound rabbit Abs were detected
using peroxidase-labeled goat anti-rabbit IgG (DakoCytomation). Ch14.18
served as a positive control and was developed by a peroxidase-labeled,
anti-human Fab (Pierce, via KMF).
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Numerous clinical trials using Ab2␤ as an antitumor vaccine
were performed or are currently underway, investigating Ab3 development and clinical remissions (35– 41). Interestingly, in contrast to some of the above studies (22, 23, 41– 45), Cheung et al.
(46) found that high HAMA levels prevented Ab3 formation in
neuroblastoma patients treated with anti-GD2 mAbs. Compiling
results from previous studies they found the following: 1) patients
with more intensive chemotherapy pretreatment before immunotherapy had lower and usually only transient HAMA/Ab2-levels
than others (20); 2) Ab3 production, starting ⬃6 –14 mo after
mAb-therapy and persisting over years, was positively correlated
with improved outcome, i.e., prolonged event-free survival, and
was more pronounced in patients with lower and only transient
HAMA/Ab2-levels (46); and 3) high HAMA concentrations were
counterproductive for survival, as they limited efficacy of further
treatment cycles. In conclusion, Cheung et al. speculated that
heavy chemotherapy eradicated lymphoid structures, as reflected
by low HAMA/Ab2-responses, and eliminated suppressor T cell
pathways and B cells. Ab2 might then bias the recovering immune
repertoire toward the GD2 network (20, 46 – 48).
Moreover, Cheung et al. (46, 49) assumed that high Ab2 concentrations were not inductive for Ab3. In the murine model only IgM led
to Ab3 production, and IgG was rather suppressive. In a healthy
immune system, dominant B cell clones (Ab2) may prevent an
anti-idiotypic response (Ab3) against themselves by an early Ig class
switch from immunogenic IgM to suppressive IgG class before
anti-idiotypic B cells (anti-Ab2) are activated.
Currently two Ab2␤-vaccines are used for the treatment of GD2positive malignancies, i.e., 1A7, a murine Ab2␤ against 14G2a, and
4B5, created by murine/human heteromyeloma technology and directed against 14G2a as well (36, 50 –53). Proof of principle was
provided by all studies, i.e., clinical responses and Ab3 serum levels
in melanoma and neuroblastoma patients treated with 1A7, and
positive Ab3 levels in animals treated with 4B5 (50, 52, 53).
However, although Foon et al. (36) did find Ab3 IgM in some of
their melanoma patients, in general there was a positive correlation
between IgM and IgG levels of Ab3, and isolated high Ab3 IgM
levels were accompanied by disease progression (50). Because the
hypotheses and experimental findings presented above are partially
contradictory, Cheung et al. (20) suggested early on that more insight
into the activation of the idiotypic network should be obtained by
cloning such anti-idiotypic Abs directly at the B cell level; instead,
most of the clinical studies that speculated on an activation of the
idiotypic network were driven by Ab2 and Ab3 levels detected in the
sera of patients treated with either Ab1 or Ab2.
In the present study we report a set of 40 human anti-idiotypic Abs
against ch14.18, cloned for the first time directly from B cells of a
patient after ch14.18-treatment by Ab phage display technology. Two
Ab2 clones, which were investigated further, elicited an Ab3 response
in rabbits, and at least one may be suitable as a tumor vaccine for
GD2-positive malignancies. To the best of our knowledge, this work
provides the first analysis of the molecular origin of the Ig repertoire
of an anti-idiotypic immune response in an Ab-treated cancer patient.
HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE
The Journal of Immunology
RNA preparation and library construction
Four Ab phage display libraries, IgG1 ␬, IgG1 ␭, IgG2 ␬, and IgG2 ␭, were
constructed using the PBLs of patient 1, collected 30 days after his fifth
ch14.18 treatment according to published protocols (57, 58) with minor
modifications (59, 60).
Biopanning procedure on 14G2a and ch14.18
glycine, 10% methanol blotting buffer. After protein transfer, the membranes were blocked overnight with dried milk powder (Nestlé) in TTBS
consisting of 10 mM Tris-Cl (pH 7.6), 9 g/L NaCl, and 0.1% Tween 20.
After washing the membranes with TTBS, they were incubated with 4 ␮l
of biotinylated ch14.18 (5 mg/ml) in 10 ml of TTBS for 1 h, washed, and
incubated again with 1 ␮l of HRP-streptavidin in 10 ml of TTBS for another hour, or they were incubated with GK8 Fab phage, GK2 Fab phage,
and anti-BSA Fab phage (A. Osei and P. Fischer, unpublished data), respectively, and counterstained with anti-M13-HRP Ab (Amersham Biosciences). After finial washing, the membranes were developed using
3,3⬘,5,5⬘-tetramethylbenzidine membrane peroxidase substrate (Kirkegaard & Perry Laboratories).
Determination of anti-idiotypic properties of selected Fab phage
ELISA plates were coated with either ch14.18 (300 ng/well), 14G2a (250
ng/well), or OKT3 (150 ng/well) and incubated with individual phage supernatants from single colonies grown in a 48-well plate for the first
screening. In subsequent assays, 1012 phage/well for low or 1010 phage/
well for high-affinity clones, respectively, were used. Bound phages were
counterstained with anti-M13-HRP Ab (Amersham Biosciences).
Analysis of nucleotide and amino acid sequences of Ab2-Fab
Single clones picked from panned libraries and specifically bound to ch14.18
and 14G2a in initial ELISAs were further analyzed. DNA of these clones was
prepared as described above or by Fischer et al. (61). The V regions of selected
Fabs were determined from phage or plasmid DNA purified with either
Midiprep (Qiagen) or Wizard columns (Promega). Sequencing was done in an
automatic sequencer (LI-COR) using the cycle sequencing kit (Amersham
Biosciences) and infrared fluorophore (IRD41)-labeled primers. The infrared
dye-labeled sequencing primers were PelB and SeqGb for the H chains and
OmpA and SeqLb or SeqKb for the L chains (57, 63, 64). V-BASE (version
16.12; 1997) (65) was searched via the internet (vbase.mrc-cpe.cam.ac.uk)
using the program DNA-Plot (version 2.0.1; developed by W. Müller and
H.-H. Althaus, University of Cologne, Cologne, Germany) for the determination of germline segments and mutations.
Large-scale production of Fabs in E. coli
Vaccination of rabbits with Ab2-Fab and Fab phage
To remove the gene III fragment for Fab expression in E. coli (58), purified
DNA (Qiagen) of selected clones was double restricted with NheI and SpeI
in buffer M (Roche), extracted with phenol/chloroform, and purified on a
low-melting agarose gel (Biozym). The agarose was melted at 60°C, and
the vector was religated using ligase buffer and ligase (Roche) at 18°C
overnight. Large-scale production of Fabs was performed by growing a
culture of a selected clone in 20 ml of super broth medium containing 50
␮g/ml carbenicillin overnight, followed by transfer into 0.5 L of super
broth medium with 50 ␮g/ml carbenicillin and 10 mM MgCl2 and growth
allowance until an OD600 of 1.2 was reached. After the addition of 1 ml of
500 mM isopropyl ␤-D-thiogalactoside, the mixture was placed on a shaker
overnight at 30°C. The bacteria were then pelleted for 20 min at 1500 ⫻
g, resuspended in 20 ml PBS/PMSF, and disrupted by sonication for 2 min
at 1-second intervals in an ice bucket. The debris was pelleted twice at
1,500 ⫻ g and 27,000 ⫻ g at 4°C, respectively, and the supernatants were
sterile filtrated before transferring them to the affinity column.
Animal experiments were conducted by Biomed Research. Two separate
experimental settings were assessed. In group 1 two animals were immunized
intradermally with 200 ␮g of purified GK2 Fab (rabbit 1) and GK8 Fab (rabbit
2) in PBS using CFA. The animals were fortnightly boosted subcutaneously
with 100 ␮g of Fab in IFA. In group 2, three animals were immunized with
GK2 Fab phage, GK8 Fab phage, and anti-BSA Fab phage, respectively.
Phages (5 ⫻ 1013) were used for each injection. No additional adjuvant was
applied. The animals were also boosted fortnightly.
Serum samples were obtained before the first immunization and every two
weeks thereafter, until day 42. The final bleed took place after 3 months.
Purification of selected Fabs from E. coli cultures
Two milligrams of goat anti-human Fab (Sigma-Aldrich) was immobilized on
a protein G-Sepharose affinity chromatography column (Amersham Biosciences) following the manufacturer’s instructions. For purification using the
Amersham Biosciences fast performance liquid chromatography system, the
column was loaded twice with 2 ml of E. coli supernatant prepared as
described above, washed with PBS, and eluted with 0.05 M citric acid and 0.5
M NaCl (pH 2.5). Eluted fractions were immediately neutralized with 1 M Tris
buffer (pH 9) and checked for purity by silver staining using SDS-PAGE and
the PhastSystem (Amersham Biosciences). Protein concentration was determined with a bicinchoninic acid kit (Pierce). For rebuffering the samples in
PBS, PD5 or PD10 columns (Amersham Biosciences) were used according to
the manufacturer’s instructions.
Western blot: reactivity of purified Fab with ch14.18
SDS-Page (8%) was loaded with 1.5 ␮g of fast-performance liquid chromatography-purified Fabs, pooled human Fabs (Jackson ImmunoResearch
Laboratories) as controls, and a protein marker (Fig. 5A); or ch14.18,
14G2a, 138H11 (62), and anti-human Fab Ab (Fig. 5B). Gels were stained
with Coomassie blue (Bio-Rad). Blotting was performed using a polyvinylidene difluoride membrane (Hybond; Millipore) in a Trans-Blot transfer
cell (Bio-Rad) at 80 V for 1 h at 4°C in a 15 mM Tris-base, 120 mM
Results
Selection of patient 1 and characterization of his anti-variable
region Abs
A total of 66 serum samples from nine patients, drawn at various
time points during ch14.18-treatment, were available for testing.
Samples had been obtained as early as day 1 after the first ch14.18treatment up until day 275 following the sixth Ab therapy. Sera of
three of nine patients (patients 1, 6, and 8) contained anti-14G2a
variable region Abs of various levels (initial assay not shown).
None of the serum samples tested were reactive with OKT3, an Ab
with the same framework region as 14G2a. Binding to 14G2a, but
not to OKT3, was a condition (but not proof) for containing Ab2␤
internal image Abs. Serum IgG concentrations were measured before
the assessment of 14G2a and OKT3 reactivity to exclude possible
artifacts that may arise simply from very high IgG concentrations.
Serum of patient 1 revealed significant anti-variable region Ab
reactivity at all IgG concentrations as depicted in (Fig. 1a). However, even at high protein concentrations it did not bind to OKT3
(Fig. 1b). Antivariable region Abs in patient 1 showed a clear
increase after repetitive ch14.18-treatments, indicating a booster
effect (data not shown).
Abs interfering with the paratope, such as internal image Abs to
ch14.18 (Ab2␤) or Ab2␥, are required to inhibit binding of
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To increase the number of different Fab phages at the beginning of the
biopanning, all four libraries were produced separately and mixed (IgG1 ␬
together with IgG2 ␬ and IgG1 ␭ together with IgG2 ␭) just before the first
biopanning cycle. The 14G2a/ch14.18-binding Fab phages were selected
by the panning of ⬃1012 recombinant phages in Maxisorp immunotubes
(Nunc) coated with 300 ␮l of 14G2a or ch14.18 at a concentration of 30
␮g/ml PBS and blocked with 1% casein. Unbound phage was removed,
and the tubes were washed vigorously up to 10 times with 0.2% casein in
PBS. Then, the bound phages were eluted with 300 ␮l of 0.1 M HCl/
glycine (pH 2.2) and neutralized with 60 ␮l of 2 M Tris-HCl (pH 9). Eluted
phages were used to infect 3 ml of a fresh Escherichia coli XL1-Blue
culture grown with tetracycline. After a 20-min incubation at 37°C on a
shaker at 240 rpm, the number of eluted phages was determined by plating
dilutions on carbenicillin (100 ␮g/ml) plates. Transformed bacteria were
diluted in 10 ml super broth (57, 60) supplemented with 10 mM MgCl2 and
20 ␮g/ml carbenicillin, grown for 1 h as described above, and then further
diluted in 100 ml of super broth with 50 ␮g/ml carbenicillin. After a 1-h
incubation, 1011 VCSM13 helper phages were added. Following another
2-h incubation kanamycin was added, and the culture was grown overnight.
Panning on 14G2a/ch14.18 was repeated three more times (59, 60).
After the last panning step, single colonies were used to grow recombinant Fab phage in 48-well plates in 300 ␮l of super broth containing 50
␮g/ml carbenicillin and 10 mM MgCl2. Diluted VCSM helper phages were
added after 5.5 h at 37°C on a shaker. After an additional hour of incubation kanamycin was added, and the cultures were grown overnight. Phage
supernatants for initial ELISA were collected after a 30-min centrifugation
at 2000 rpm using a plate rotor. The resulting pellets were used for analytical DNA preparations as described (61).
7777
7778
HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE
chains, and both the ␬ and ␭ L chains had been used (data not
shown).
Library sizes, i.e., the maximum number of different clones, of
the four libraries constructed were 9.1 ⫻ 106 for IgG1 ␬, 3.4 ⫻ 106
for IgG1 ␭, 6.2 ⫻ 106 for IgG2 ␬, and 6.7 ⫻ 106 for IgG2 ␭. The
presence of complete H and L chain inserts was analyzed in 10
clones randomly picked per library; 100% of the IgG1 ␬, 70% of
the IgG1 ␭, 70% of the IgG2 ␬, and 60% of the IgG2 ␭ library
contained both inserts as estimated by DNA-restriction from single
colonies and also from total phagemid DNA.
FIGURE 1. Patients’sera were tested for the presence of anti-idiotypic
Abs following anti-GD2 therapy with ch14.18. Serum samples had been
drawn on day 30 after the fifth ch14.18 therapy treatment of neuroblastoma
patient 1, 15 days after the fourth ch14.18 treatment of patient 6, and 21
days after the fifth treatment of patient 8. Serum from two healthy controls,
control 1 and control 2, served as a negative control, and an anti-mouse IgG
served as a positive control (ps. and pos.). a, Microtiter plates were coated
with 14G2a. Only sera of patients that showed reactivity in a preliminary
assay (not shown) were tested here again for antivariable region reactivity,
i.e., the presence of Ab2. The serum of patient 1 demonstrated strong
reactivity with 14G2a, the murine relative of ch14.18, at all dilutions assessed, whereas neuroblastoma patients 6 and 8 showed only weak reactivity at high IgG concentrations, similar to the healthy controls. b, Antiframework reactivity of the serum of patient 1 was excluded by binding
assessment on OKT3-coated plates. Only at high IgG concentrations did
the serum of patient 1 demonstrate weak binding.
ch14.18 to target Ag GD2. The serum of patient 1 completely
inhibited GD2 binding of ch14.18 at dilutions of up to 1/256,
whereas the serum of another neuroblastoma patient showed only
some GD2 binding inhibition at low serum dilutions of up to 1/4.
Serum of a normal donor did not inhibit binding at all (Fig. 2).
These results were a further indication (but still not proof) that the
serum of patient 1 may contain internal image Abs (Ab2␤) aiming
at the binding site of ch14.18.
Combinatorial phage display libraries
To construct specific Ab phage display libraries, the anti-idiotypic
Abs of patient 1 were further characterized in ELISAs. Abs were
exclusively of IgG isotype, IgG1 and IgG2 subclass for the H
Biopanning of phage
Four biopannings were performed. Corresponding ␬ and ␭ libraries
were mixed. The IgG1 ␬ library, together with the IgG2 ␬ library,
was selected on either ch14.18 (panning I) or 14G2a (panning II);
the IgG1 ␭ and IgG2 ␭ libraries were panned on either ch14.18
(panning III) or 14G2a (panning IV).
Panning I and II showed a continuous strong increase of specifically binding phage during all four cycles of biopanning. The
phage output/input ratio before and after incubation of the library
with the Ag increased from 6.9 ⫻ 10⫺4 % (panning I) and 4.6 ⫻
10⫺4 % (panning II) after the first incubation to 1.1% (panning I)
and 1.5% (panning II) after the fourth round, respectively. However, similar clear continuous increases of phage titers could not be
demonstrated for pannings III and IV. Specific phage output/input
ratios ranged between 4.0 ⫻ 10⫺6 and 2.3 ⫻ 10⫺3 % for panning
III and between 0.7 ⫻ 10⫺6 and 2.3 ⫻ 10⫺3 % for panning IV.
After the final panning, 44 single clones of Fab phage were grown
from each panning in 48-well plates and analyzed for reactivity
with ch14.18 and 14G2a in ELISA. After restriction digestion with
frequent-cutting BstN1, indicating some genetic variety, 10 positive clones from each of the four experiments were sequenced.
Characterization of selected clones
Sequencing revealed at least 14 clones with different DNA sequences among the 40 picked clones. One clone lacked a L chain
and was not further analyzed. Importantly, one clone (GK8) was
isolated as often as 19 times. All 13 different clones proved to have
anti-idiotypic properties by binding to both ch14.18 and 14G2a in
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Binding inhibition of ch14.18 to GD2 by serum of patient 1
and two controls (a neuroblastoma patient and a negative (Neg.) control).
Depicted is the percentage of binding inhibition as determined by B/Bo,
where B is absorbance with inhibitor and Bo is absorbance without inhibitor. Bo was set at measurement endpoint OD of 2.5. Serum from patient
1 completely inhibited binding of ch14.18 to GD2. Serum from a healthy
donor served as a negative control. Serum from a neuroblastoma patient,
who had been treated with ch14.18 at another institution and therefore was
not included in this study, showed very little binding inhibition..
The Journal of Immunology
FIGURE 3. Assessment of the binding specificity of 12 Fab phage
clones to ch14.18 and 14G2a. Fab phage representatives of the 12 clones
with different sequences were retested for their capacity to bind both 14G2a
and ch14.18, as well as for their lack of reactivity with OKT3, to confirm
that all clones bind within the variable region of ch14.18 and 14G2a. Clone
T14-5, not tested in this assay, and the weakly reactive TCH-9 were determined to be positive in the previous ELISA (not shown). For clones that
initially showed very strong reactivity with ch14.18 and 14G2a, concentrations of 1010 phages per well were used; otherwise, 1012 phages per well
were used. Anti-BSA-Fab phage, Fab phage selected with IVIG (LO31),
phage containing the empty vector, and PBS were used as negative controls.
An anti-mouse-antibody was used as a positive (pos) control for 14G2a and
OKT3 as well as an anti-human Fc Ab for ch14.18. All tested clones bound
both ch14.18 and 14G2a and, therefore, were considered anti-idiotypic Abs.
be noted that H chain CDR3, which normally participates strongest
in Ag binding, cannot be included in this analysis because it is
composed of irregular VDJ, P/N nucleotide addition, irregular
frame shifts, and inverse D combinations that occur before Ag
selection. The results presented above relate to 10 different clones
among 36 evaluable, anti-idiotypic Ab clones. Considering that
several clones appeared as frequently as 19 times among the 36
analyzed clones, their significance is clearly increased.
Anti-idiotypic properties of GK2 and GK8
Anti-idiotypic binding properties of both Fabs and the corresponding Fab phages of GK2 and GK8 following large scale production
and purification are depicted in Western blot analyses (Fig. 5, a
and b). GK2 and GK8, both primarily selected by biopanning on
murine 14G2a, also bind specifically to chimeric ch14.18 and,
thus, are anti-idiotypic Abs.
Secondly, inhibition of ch14.18 binding to GD2 could be demonstrated for the isolated Fabs. Concentrations as low as 1.6 ␮g of
GK2 Fab and 0.8 ␮g of GK8 Fab were able to inhibit binding of
ch14.18 to GD2 by 70% or completely, respectively. An anti-BSA
Fab, used as a control, showed no competition (Fig. 6). This confirmed that GK2 and GK8 are anti-idiotypic Abs of the type Ab2␤
or Ab2␥.
GK8 recognized and bound to the same epitope as the natural
anti-idiotypic serum Abs of patient 1 did. At a concentration of 2.8
␮g, GK8 Fab suppressed binding of diluted (1/20,000) patient serum
to 14G2a by 84%. GK2 Fab completely lacked this capacity (Fig. 7).
Proof that they are true internal image Abs (Ab2␤) of GD2 was
found for GK8 Fab and, to a much lesser extent, for GK2 Fab
when they induced anti-GD2 Abs (Ab3) in the xenogeneic rabbit
system. Preimmune rabbit sera were tested for positive reactivity
with GK2 and GK8 Fabs and the corresponding Fab phages (data
not shown) and for positive GD2 binding to preclude presence of
Ab3 before vaccination. All rabbit sera demonstrated a very strong
reactivity to their respective vaccine in ELISA 42 days after immunization, including the anti-BSA control (not shown).
After immunization, rabbits of group 1, immunized with GK2
Fab and GK8 Fab, demonstrated an increase of anti-GD2 titers
between day 0 and day 63 after initial vaccination, which was more
pronounced for GK8 Fab (Fig. 8a). Samples were adjusted for
their IgG content to account for variable IgG levels in different
rabbits before and after immunization. Vaccination of rabbits of
group 2, which received GK2 and GK8 Fab phage, again demonstrated positive anti-GD2 responses (Ab3) after treatment. In contrast, a control immunized with the anti-BSA Fab phage remained
negative on GD2. Samples of group 2 were only available until day
42 after initial immunization (Fig. 8b). In summary, GK2 and GK8
were able to induce an anti-GD2 Ab response in both experimental
settings, which was very weak for GD2 but strong for GK8. Thus,
at least GK8 can be termed a true Ab2␤.
As expected, the use of human Fabs as a vaccine in rabbits also
elicited immune responses against the framework region of the
injected human fragment. The background of interfering Abs could
be reduced by preincubation with IVIG. Without preincubation, a
2.98-fold increase for GD2-binding Abs against GK8 Fab phage
was observed upon comparison of prevaccination and postvaccination samples. Preincubation with IVIG resulted in a 4.06-fold
increase of specific GD2-binding Abs (data not shown).
Discussion
Phage display technology enabled the cloning of a repertoire of
anti-idiotypic Abs (Ab2) from B lymphocytes of a tumor patient
after passive immunotherapy with chimeric anti-GD2 Ab ch14.18,
designated Ab1 in this case. Upon repetitive selection of Fab phage
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
repetitive ELISAs (Fig. 3). Three clones (T14 –10, TCH-1, and
TCH-2) provided problems in sequence analysis and are not listed
in the tables and sequence alignments (Fig. 4). Use of H and L
chains and amino acid sequences of CDRs varied greatly. Amino
acid sequences revealed sequence identities between 26.8 and
100% among the H chains of the selected clones (Fig. 4a) and
between 28.5 and 100% among the L chains of the selected clones
(Fig. 4b). Identical sequences are only shown once in the figures.
Table I shows the corresponding germline gene origins for the
different Ab chain sequences. Identities to eight different VH germline genes were between 94.9 and 100%. Three different VH families were used. Seven of 10 different clones belonged to the VH3,
2 of 10 to the VH1, and 1 of 10 to the VH4 family. Fifty percent of
all VH sequences originated from the most common 3-30 (3 of 10
clones) or 3-23 (2 of 10 clones) germline gene loci. DH and JH
sequences could be aligned to all 10 clones. However, as indicated
in Table I, several DH sequences had to be aligned manually because of weak homology scores from V-BASE, leaving open several possibilities. JH4b was found in 8 of 10 clones.
Ab L chains included V␭1, V␭6, V␬1, and V␬3 chains. Five of 10
clones had a ␭ L chain predominantly belonging to the L1 family (4
of 10). The ␬ L chains used belonged to the K1 (3 of 10) and K3 (2
of 10) L chain families with almost equal spread. Identities to their
putative VL germline genes ranged between 93.90 and 99.60%.
Mutation rates of selected clones in relation to corresponding
VH/L germline sequences are shown in Table II. Remarkably, in
the framework region not 1 of 10 clones showed a replacementto-silent mutation (R:S) ratio of ⬎2.9 for the H chain, whereas 6
of 10 did for the L chain. With regard to CDRs 1 and 2, 6 of 10
clones revealed an R:S ratio of ⬎2.9 for the H chain, and 6 of 10
did for the L chain CDRs. CDRs 1 and 2 contained no replacement
mutations in 3 of 10 H chains and 1 of 10 L chains. An R:S ratio
of ⬎2.9 in 60% of all H chain CDRs and 60% of the L chain CDRs
may be indicative of Ag-driven Ab selection by ch14.18. It should
7779
7780
HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. Alignment of 10 different, complete amino acid (aa) sequences. Shaded areas mark amino acid identities among the clones. a, Alignment
of amino acid sequences of Ab H chain variable regions. According to the Kabat nomenclature (91) CDR1, CDR2, and CDR3 correspond to the amino
acid sequences 32–37, 52– 68, and 101–122, respectively, in this figure. b, Alignment of amino acid sequences of Ab L chain variable regions. According
to the Kabat nomenclature (91) CDR1, CDR2, and CDR3 correspond to the amino acid sequences 22–34, 50 –56, and 91–101, respectively, in this figure.
GenBank accession no. AM259131-AM259150.
display libraries on the target Ag ch14.18 and its murine relative
14G2a, positive binders were enriched. The selected Ab2 clones
GK2 and GK8 as well as another 38 Fab phage clones demonstrated strong reactivity with both ch14.18 and 14G2a. This was
repeatedly confirmed (Fig. 3). Specificity and selectivity of binding was confirmed in Western blot analysis (Fig. 5, a and b). Both
anti-idiotypic clones inhibited binding of ch14.18 to the tumorassociated Ag GD2 (Fig. 6). Surprisingly, GK8 Fab alone was able
to inhibit binding of all of the anti-idiotypic Abs of patient 1 by
84%, suggesting that it is identical with the majority of the patient’s original anti-idiotypic Abs (Fig. 7) (66). Immunization of
rabbits with either soluble GK2 or GK8 Fab or their Fab phages
produced a continuous rise in Ab3 serum levels (Ab3⬘) in these
animals, as indicated by increased GD2 binding (Fig. 8, a and b).
The cloned human anti-idiotypic Fab GK8 may be suitable as a
tumor vaccine.
The Journal of Immunology
7781
Table I. Putative germline gene sequences of 14G2a/Ch14.18 selected clones
Clone
VH
(Frequency) Family
Homology
VH
Locus
(%)
VH Germline Genea
DH a
JHa
VL/K
Family
VL/K Germline Genea
VL/K
Locus
Homology
(%)
JL/Ka
3
DP51b
Z12351
3-48
99.93
3-9b
X97051
JH4b
X86355
L1
1c.10.2/DPL2 . . .
Z73654
b
1c
98.50
JL3b
D87017
T14-5
(1)
3
DP51b
Z12351
3-48
97.40
3-9b
X97051
JH4b
X86355
L1
1c.10.2/DPL2 . . .
Z73654
b
1c
98.50
JL3b
D87017
T14-7
(6⫻)
3
DP-46/3d216 . . .
Z12346
b
3-30.3
94.90
5-05/5-18/3-16c JH4b
X97051
X86355
K3
L16/humkv31esc
X72815
L16
93.90
JK1
J00242
T14-9
(1)
3
DP-47/V3-23 . . .
Z12347
b
3-23
98.90
7-27
X97051
JH4b
X86355
K3
DPK22/A27 . . .
X93639
A27
98.90
JK2
J00242
TCH-3
(1)
3
COS-8/hv3005f3 . . .
Z17394
3-30
96.70
3-22
X97051
JH4b
X86355
L1
1b.366F5/DPL5 . . .
Z73661
b
1b
99.60
JL2/3
M15641
TCH-5
(1)
1
DP-10/hv1051 . . .
Z12312
1-69
96.00
3-9b
X97051
JH4b
X86355
L6
6a.366F5/V1-22 . . .
Z73673
b
6a
99.30
JL3
D87017
TCH-6
(1)
1
1-03
100.00
3-03
X97051
JH4b
X86355
L1
1b.366F5/DPL5 . . .
Z73661
b
1b
99.60
JL2/3
M15641
TCH-8
(1)
3
DP-47/V3-23 . . .
Z12347
3-23
96.30
3-22/5-12c
X97051
JH6c
K1
DPK9/O12b
Z93627
O12/O2
94.30
JK1
J00242
GK-2
(1)
3
DP-49/1.9III . . .
Z12349
3-30
3-30.5
98.90
K1
DPK5/Vbb
X93623
L5
97.60
JK1
J00242
GK-8
(19⫻)
4
DP-70.4d68 . . .
Z12370
4-04
96.40
K1
DPK9/O12b
Z93627
O12/O2
98.50
JK1
J00242
b
DP-25/VI-3bb
Z12327
b
b
b
6-19/1-26/6-06c JH4b
X97051
X86355
7-27
X97051
JH3b
X86355
b
a
Numbers in italics reveal EMBL/GenBank accession numbers, with sequence nomenclature according to VBASE (vbase.mrc-cpe.cam.ac.uk).
Indicates mapped germline sequence.
c
Several D or V␬ germline sequences possible, respectively.
b
This approach allowed the characterization of the molecular origin of totally human anti-idiotypic Abs cloned from a previously
treated neuroblastoma patient. To the best of our knowledge, this
is the first time that numerous totally human Ab2s have been directly cloned from a tumor patient’s B cell repertoire, permitting
deeper insight into the Ig gene usage within the anti-idiotypic Ab
repertoire.
Screening neuroblastoma patients for Ab2-serum levels after
ch14.18 treatment to choose a suitable B cell donor demonstrated that,
in our single center study, among the 65 serum samples of nine
patients analyzed, only three patients produced a significant immune
response against the variable region of ch14.18 (Ab2). These three
patients are still alive without signs of disease for 4.5–7 years and
have been off treatment for 4 –5.5 years. Regardless of Ab2 serum
reactivity, no Ab3 could be measured in patients’ sera available for
testing. However, although first Ab3 responses had been reported as
early as 6 mo after initial Ab1-treatment, time intervals of 12 mo
following initial ch14.18 treatment (allowing for regular blood draws
according to therapy protocol NB97 of the Society for Pediatric
Oncology and Hematology in our case) may still have been too short
(46, 54). Survival analysis of the remaining six patients shows two
patients in complete remission for 5 and 7 years, respectively. Two
patients relapsed, one with stable disease after surgery and radiotherapy and the other with progressive chronic neuroblastoma, and two
patients died of progressive disease (67).
Discrepancies between the studies of Yu et al. (14), Cheung et
al. (30), and Ozkaynak et al. (68) and our results are striking in
respect to anti-GD2 immune responses. Whereas they had a high
frequency of immune responses to ch14.18 and 3F8, we only detected an immune response to ch14.18 in three of nine patients (14,
20, 68). Neither of the above studies asked for precautionary routine administration of corticosteroids to preclude anaphylactic re-
activity to mAb ch14.18 as did our protocol NB97 (14, 54, 68).
Corticosteroid premedication at least partially counteracts the initiation of a natural immune response needed for the activation of
the idiotypic network. Applying 47 courses of ch14.18 treatment to
nine patients, we rarely observed any side effects, in agreement
with the overall results of our NB97 multicenter study (69). Interestingly, patient 1, who was the only one in our patient group not
pretreated with corticosteroids and who suffered from ch14.18 side
effects such as severe hyperthermia, pain, and transient renal tubulopathy during/following his first course of ch14.18 therapy, developed significant Ab2 levels against ch14.18 and is still in remission (Fig. 1a). According to Simon et al. (69), in the NB97 trial
corticosteroids were given to 62 of 151 patients in 175 of 695
evaluable ch14.18 courses. No differences in event-free survival
and overall survival were found with regard to corticosteroids.
However, development of anti-idiotypic Abs was not evaluated.
Anti-idiotypic Abs were and are used as surrogate tumor-associated Ags in numerous vaccination trials, producing clinical remissions and confirming the idiotypic network. Until now, for the
most part murine and other xenogeneic anti-idiotypic Abs, partially humanized or genetically modified, were cloned for tumor
therapy. A vaccination trial for GD2-positive neuroblastoma using
a murine anti-idiotypic 1A7 to 14G2a proved this treatment to be
well tolerated. Although all 31 patients developed Ab3 (anti-1A7),
GD2-binding was only seen in five of seven patients treated. Because of the xenogeneic murine protein used as vaccine, much of
the immune response induced was directed toward the murine
framework, whereas specific Ab3 responses were low (53, 70).
The latter results parallel those of our rabbit vaccination experiments, which showed significant immune responses toward the
human framework and constant regions of GK2 and GK8, resulting in higher ELISA background when sera were not preadsorbed
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
T14-1
(4⫻)
7782
HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE
Table II. Mutation rates in 14G2a/Ch14.18 selected VH and VL
VH CDRs 1, 2
Clone
T14-1
T14-5
T14-7
T14-9
TCH-3
TCH-5
TCH-6
TCH-8
GK8
1/66
1/0
1/66
1/0
4/66
4/0
0/66
0
4/66
3/1
7/66
5/2
0/66
0
2/66
2/0
0/66
0
3/66
3/0
Percentage
Ratioc
No./Total
R/Sb
1.5
⬁d
1.5
⬁d
6.1
⬁d
0
0
6.1
3
10.6
2.5
0
0
3.0
⬁d
0
0
4.5
⬁d
1/207
0/1
6/207
3/3
10/207
7/3
3/207
1/2
5/204
3/2
4/208
2/2
0/205
0
8/207
4/4
3/207
1/2
7/208
5/2
a
Percentage
Ratioc
0.5
0
3.9
1
4.8
2.3
1.4
0.5
2.5
1.5
1.9
1
0
0
3.9
1
1.4
0.5
3.4
2.5
Entire Region
VL CDRs 1, 2, (3)
No./Totala
Percentage
2/273
0.07
7/273
2.6
14/273
5.1
3/273
1.1
9/270
3.3
11/274
4.0
0/271
0
10/273
3.7
3/273
1.1
10/274
3.6
No./Total
R/Sb
3/87
2/1
3/87
2/1
7/75
6/1
2/81
2/0
1/87
0/1
1/81
1/0
1/72
1/0
5/75
4/1
2/63
2/0
3/75
2/1
a
VL Framework Regions
1–3
Percentage
Ratioc
No./Total
R/Sb
3.4
2
3.4
2
9.3
6
2.5
⬁d
1.1
0
1.2
⬁d
1.4
⬁d
6.7
4
3.2
⬁d
4.0
2
1/183
1/0
1/183
1/0
9/186
7/2
1/186
1/0
0/177
0
1/189
1/0
0/183
0
10/186
5/5
4/186
0/4
1/186
1/0
a
Percentage
Ratioc
0.5
⬁d
0.5
⬁d
4.8
3.5
0.5
⬁d
0
0
0.5
⬁d
0
0
5.4
1
2.2
0
0.5
⬁d
Entire Region
No./Totala
Percentage
4/270
1.5
4/270
1.5
16/261
6.1
3/267
1.1
1/264
0.4
2/270
0.7
1/255
0.4
15/261
5.7
6/249
2.4
4/261
1.5
a
No./Total means number of mutations/number of all bases in the region.
R/S represents number of replacement/silent mutations.
c
Ratio stands for R/S ratio.
d
⬁ denotes infinity, although mathematically not correct.
b
with pooled human IgG. Similar adsorption experiments demonstrated an Ab3 response, i.e., positive GD2 binding in 40 of 47
melanoma patients treated with 1A7 (36).
When human anti-idiotypic Ab 105AD7, produced by the fusion
of a human/mouse heteromyeloma and B cells of a patient treated
with ␣791T/36 (a mouse Ab directed against gp72), was applied to
osteosarcoma patients, 11 of 28 patients had an Ab3 response (38,
71). Fusion cell lines of human/mouse heteromyelomas, however,
frequently are not stable and contain murine glycosylation (72–
75). Use of a completely human vaccine in the human system
should therefore be advantageous and even more effective by overcoming problems of nonspecific immunoreactivity. A possible alternative may also be peptide mimotopes of GD2 (76, 77).
Goletz et al. (78) described the selection of large diversities of
single-chain variable fragment (scFv) anti-idiotypic Abs from
three large naive human phagemid libraries (Griffin I and Tomlinson I ⫹ J libraries). They accomplished an increase of antiidiotypic scFv binders by improving elution and selection procedures of scFv. For the glycine elution, which was also used in our
work, their yield of specifically binding anti-idiotypic scFv was 5
of 96 (5%), whereas our study used an “immunized” library in
which the B cell donor had been previously stimulated with the
target Ag (mAb ch14.18), and at least 22% anti-idiotypic clones
were selected.
After each panning 44 clones were analyzed for binding to
14G2a, making 176 clones in total. Of these, 40 clearly positive
clones (22,7%) could be selected and further characterized according to their gene sequence. Thirty-nine of forty clones were complete Fabs, with binding to ch14.18 and 14G2a. One clone did not
contain a L chain and was not further analyzed. Interestingly,
many clones had identical DNA sequences, and only 13 were different. In contrast, anti-idiotypic scFv from a nonimmunized library (78) showed a greater sequence diversity of 70%.
A possible artificial new combination of H and L chains during
library preparation cannot be excluded and has been discussed in
detail earlier (66, 79 – 82). However, in immunized donors (such as
an Ab-treated patient), the frequency of Ag-specific B cells is very
high (83, 84). During panning with a monovalent phage system
such as pComb3H, phages with the highest affinity are selected,
requiring a H and L chain combination already optimized in the
patient. This is reflected in the frequent selection of GK8, which
demonstrated excellent anti-idiotypic properties and induced Ab3
in rabbits. GK8 appeared as frequently as 19 times among 40 sequenced clones, always with the same combination of H and L
chains. This finding, together with the conclusions from Fig. 7,
argues for the original combination at least in this clone. It may be
different for GK2. Similar conclusions were drawn in experiments
by Rapoport and coworkers (66, 79, 80) with anti-thyroid peroxidase Abs. Similarly as in our inhibition experiment (Fig. 7), a
combination of only two cloned Fabs was able to inhibit the binding of patient’s serum autoantibodies to the Ag thyroid peroxidase.
In subsequent shuffling experiments, they observed that promiscuity of H and L chains was very restricted (66, 79, 80). In addition,
they selected the same H and L chain pairings in multiple patients
(85).
Three of the 13 representatives of our different clones (TCH-1,
TCH-2, and T14 –10) were excluded from further analysis due to
sequencing problems. T14-1 and T14-5 showed identical L chain
sequences. Their H chains differed in one amino acid in CDR1 as
well as CDR3 and showed two amino acid exchanges in the framework 2 region (Fig. 4, a and b). DH alignment of T14-7 was not
unequivocally possible. Candidate DH loci include 5-05, 5-18, and
3-16 (Table I). V-BASE alignment also was critical in regard to the
L chain alignment for this clone. Although the highest score was
obtained for alignment to DPK22/A27, germline sequence L16/
humkv31es demonstrated higher homology, as DPK22/A27 contained an amino acid in position 27a of CDR1, which was not part
of T14-7. Germline genes 3A7 and 3A9 in descending order also
revealed homology to the L chain of T14-7.
An analysis of the R/S ratio, giving the ratio of replacement to
silent mutations (Table II), suggested an Ag-driven selection of
anti-idiotypic B cells in the patient triggered by ch14.18-treatment.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
GK2
No./Total
R/Sb
a
VH Framework Regions
1–3
The Journal of Immunology
7783
FIGURE 5. Western blot analysis of purified GK2 and GK8 Fab (a) and
GK2 and GK8 Fab phage (b). a, SDS-Page of blotted, purified GK2 and
GK8 Fabs, and pooled human Fabs as a control is shown. Coomassie
staining (top panel) visualizes GK2 (lane 2 from left) and GK8 (lane 3 from
left), as well as human Fab (lane 4 from left) at a molecular mass of ⬃60
kDa (lane 1, protein (Prot.) marker). The bottom panel shows binding of
blotted GK2 and GK8 Fabs stained with biotinylated ch14.18. Thus, GK2
Fab and GK8 Fab, selected on 14G2a, both bind specifically to ch14.18 as
well. b, SDS-PAGE. Blotted ch14.18 (lane 1 from left), 14G2a (lane 2 from
left), 138H11, a murine IgG1 that served as a negative control (lane 3 from
left), and anti-human Fab antibody, which served as positive control (lane
4 from left), were stained with Coomassie stain (top panel), revealing a
molecular mass of ⬃180 kDa. After incubation with GK8 Fab phage (panel
2 from top), GK2 Fab phage (panel 3 from top), and anti-BSA Fab phage
(panel 4 from top), anti-M13-HRP Abs were used for detection. GK2 Fab
phage and GK8 Fab phage both demonstrated specific reactivity with
14G2a and ch14.18.
Six of ten (60%) different clones showed an R/S ratio of ⬎2.9 for
CDRs 1 and 2 of their H chains, and six of ten (60%) did so for
CDRs of their L chains. Taking into account that some clones
appeared repeatedly, 32 of 36 (89%) clones showed somatic replacement mutations in CDRs of VH regions, and 11 of 36 (31%)
did so for CDRs of their VL regions.
This result is interesting, because earlier studies on bone marrow
transplant (BMT) recipients had demonstrated that rearrangements
in BMT recipients exhibited far fewer somatic mutations than did
rearrangements from healthy subjects (86, 87). Although our B cell
donor did not undergo BMT, intensive chemotherapy is also
known to destroy lymphoid tissue. Because the failure to accumulate somatic mutations in rearranged VH genes is consistent with a
maturational arrest at a very late state of B cell differentiation, and
because somatic mutations and affinity maturations are thought to
take place in lymph node germinal centers, it is a popular hypothesis that the failure of germinal center processes prevents normal
accumulation of somatic mutations following immunization in
BMT recipients (86, 87). But, unlike studies in BMT recipients,
Ab clones picked from our “immunized” library from a heavily
pretreated neuroblastoma patient exhibit a proportion of somatic
mutations, comparable to what we and others found in B cell li-
braries of healthy subjects (60, 87). In this study, 30% of somatic
mutations were detected in the B cell population of these subjects,
divided into 10% of such mutations in preimmune B cells, 70% in
Ag-stimulated B cells, and 20% IgM mutations. In contrast, only
1–10% of somatic mutations were found in B cells of BMT recipients (87). In summary, the immunological capacity of our patient
to respond to foreign Ags seems rather comparable to that of a
healthy subject.
Further studies on BMT recipients and the reconstitution of the
Ig VH repertoire were able to describe particular patterns of VH
expression in healthy subjects and allografted/autografted patients,
the latter mimicking ontogeny of the Ig repertoire (88). Although
there is a bias due to PCR primer selection, the use of VH-genes in
10 different Ab2-clones in our current study was VH3 (70%) ⬎
VH1 (20%) ⬎ VH4 (10%). Accounting for the total numbers of
clones changed the order to VH4 (53%) ⬎ VH3 (42%) ⬎ VH1
FIGURE 7. Competition of purified Fabs with the natural anti-idiotypic
Abs of patient 1 on 14G2a. Purified GK2 and GK8 Fabs competed with the
natural anti-idiotypic Abs of patient 1, i.e., diluted patient’s serum, for
binding to 14G2a. GK8 Fab demonstrated nearly complete binding inhibition, indicating that it represents the majority of the patient’s antiidiotypic Abs. The patient’s serum had to be diluted to adjust for comparable IgG concentrations of both competitors.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Competition of purified Fabs with ch14.18 on GD2. GK2
and GK8 Fabs were purified by affinity chromatography using immobilized
anti-human Fab Abs. Competition of purified Fabs with ch14.18 on GD2
clearly showed that GK2 Fab and GK8 Fab were able to significantly
inhibit (GK2 Fab) or completely suppress binding (GK8 Fab) of ch14.18 to
GD2 even at low concentrations, in contrast with an anti-BSA-Fab (negative control). Depicted is the percentage of binding inhibition as determined by B/Bo, where B is absorbance with inhibitor and Bo is absorbance
without inhibitor. Bo was set at measurement end point OD of 2.5.
7784
HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE
Acknowledgments
FIGURE 8. Ab3 production in rabbits following immunization with
Ab2 Fab and Ab2 Fab phage. An increase of anti-GD2 antibodies in rabbits
immunized with GK2 and GK8 Fabs (a) and GK2 Fab and GK8 Fab phage
(b) is demonstrated. Sera were preincubated with human Fabs. a, Rabbit
sera from day 0 and day 63 after immunization were analyzed for GD2
binding. Comparing pretreatment and posttreatment sera, rabbits vaccinated with GK8 Fab showed a high 9.8-fold increase in GD2 binding
compared with a low 1.5-fold increase for GK2 Fab. Results represent the
mean of two measured values and were significant for GK8 (p ⫽ 0.019)
according to a paired t test (SigmaStat), but not for GK2 (p ⫽ 0.181).
Ch14.18 and PBS were not used for vaccinations but served as a positive
and a negative control, respectively, for the ELISA. Bound ch14.18 was
detected by a peroxidase-labeled anti-human IgG Fab Ab. b, Similarly,
rabbits immunized with GK8 Fab phage demonstrated a 4.1-fold increase
in anti-GD2 reactivity comparing prevaccination and postvaccination samples. Again, this result is more pronounced than what is shown for immunization with GK2 Fab phage, which yielded an increase of 2.2-fold in
anti-GD2 reactivity. For the latter group, serum samples were available
only until day 42 after initial immunization. In contrast to the anti-Id vaccinations, a control rabbit immunized with the anti-BSA Fab phage showed
no increase in binding to GD2. The data are representatives of several
independent assays with or without preadsorption on IgG that used antirabbit IgG or anti-rabbit IgG/IgA/IgM for detection. Those other assays
(not shown) yielded comparable results, although the absolute values
varied.
(6%). Whereas the relative numbers in the first order resemble the
immunological VH expression pattern of a healthy subject, the absolute numbers with a VH3 decrease and a correlative VH4 increase are suggestive of a recovering immunological status at a
state of redeveloping “self-recognition” (89). The high proportion
of ␬ L chains would also point in this direction.
Comparison of the above Ig gene selection from an “immunized” patient provides a striking contrast to previously analyzed
Ab repertoires, panned on IVIG, of a healthy individual, thrombocytopenia patients, and a systemic lupus erythematosus patient.
In this study we observed that the majority of selected Abs were
We thank Heike Lerch for excellent technical assistance, Prof. G. Gaedicke
(Charité, Berlin, Germany) and Prof. A. Yu (University of California at
San Diego, La Jolla, CA) for discussion, Prof. R. Handgretinger (St. Jude
Children’s Hospital, Memphis, TN) for providing 14G2a, and Prof. C.
Barbas (The Scripps Research Institute, La Jolla, CA) for providing the
pComb3H vector. Moreover, we thank the personnel of wards 30, 39, and
T23 Hem/Onc outpatient center (Charité Children’s Hospital) for their help
in sampling patient sera.
Disclosures
M. M. Uttenreuther-Fishcer, J. A. Krüger, and P. Fischer have applied for
a German patent, ⬙Mittel humanen Ursprungs zur Vakzination genen GD2positive Tumore.⬙
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HUMAN ANTI-IDIOTYPIC ANTIBODY VACCINE