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of June 18, 2017.
Novel Ganglioside Antigen Identified by B
Cells in Human Medullary Breast
Carcinomas: The Proof of Principle
Concerning the Tumor-Infiltrating B
Lymphocytes
Beatrix Kotlan, Peter Simsa, Jean-Luc Teillaud, Wolf
Herman Fridman, Jozsef Toth, Michael McKnight and Mark
C. Glassy
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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 © 2005 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2005; 175:2278-2285; ;
doi: 10.4049/jimmunol.175.4.2278
http://www.jimmunol.org/content/175/4/2278
The Journal of Immunology
Novel Ganglioside Antigen Identified by B Cells in Human
Medullary Breast Carcinomas: The Proof of Principle
Concerning the Tumor-Infiltrating B Lymphocytes1
Beatrix Kotlan,*§¶ Peter Simsa,* Jean-Luc Teillaud,† Wolf Herman Fridman,† Jozsef Toth,‡
Michael McKnight,§¶ and Mark C. Glassy2§¶储
R
ecombinant Abs and their fragments represent now a
very high percentage of all biological proteins undergoing clinical trials for diagnosis and therapy (1, 2). Clinical research in the area of Ab-based tumor-targeted therapy has
been driven for many years by the prospect of identifying cell
surface Ags with sufficient restrictive tissue expression patterns to
allow a selective and specific tumor tissue accumulation of the Ab
(3). Technology for Ab design has taken enormous strides forward
through new library display, and selection procedures that serve as
perfect tools for searching for tumor-specific or any other Ags
have been described (4, 5). Even with these new procedures, there
are difficulties in identifying real and reliable tumor-specific structures with characteristics that enable them to be used further as
*National Medical Center/Institute of Haematology and Immunology, Budapest,
Hungary; †Institut National de la Santé et de la Recherche Médicale Unité 255, Centre
de Recherches Biomedicales des Cordeliers, Universite Pierre et Marie Curie, Paris,
France; ‡National Institute of Oncology, Budapest, Hungary; §Rajko Medenica
Research Foundation, San Diego, CA 92121; ¶Shantha West, San Diego, CA 92121;
and 储University of California, San Diego Mechanical and Aerospace Engineering
Department, La Jolla, CA 92037
Received for publication October 6, 2004. Accepted for publication April 27, 2005.
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 project was supported by international (NATO CLG 978639) and Hungarian
grants (Eotvos XLI/34/2000), Orszagos Tudomanyos es Kutatasi Alap (National Scientific and Research Fund) (T030380), and recently by the Rajko Medenica Research
Foundation. Some earlier basic works were performed through scholarships granted
to B.K. from International Agency for Research on Cancer, Societe du Cancer,
France, bourses of French Government, and Centre International des Etudes Scientifiques, and benefited from Institut Curie, Association pour la Recherche sur le Cancer, Association pour la Recherche sur le Cancer program (C01-010), and Institut
National de la Santé et de la Recherche Médicale.
2
Address correspondence and reprint requests to Dr. Mark C. Glassy, Rajko Medenica Research Foundation, 11211 Sorrento Valley Road, Suite C, San Diego, CA
92121. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
potential targets for tumor diagnostics or therapeutics (6, 7).
Among the strategies to obtain reliable tumor target molecules, one
of the most challenging and perhaps insightful is the exploitation
of the intelligence of the natural human immune response to tumor-restricted Ags (8 –11).
The presence of B lymphocytes that infiltrate cancerous tissues
may reflect an ongoing immune response against transformed cells. In
addition, these cells may provide an interesting source for obtaining
Abs to tumor Ags, and therefore some insight into the natural anticancer immune response. Although some important earlier investigations in melanoma have been described (12–14), almost all efforts
concerning infiltrating lymphocytes were focused on T cells (15, 16).
The paucity of information concerning tumor-infiltrating B (TIL-B)3
cells has, in part, been due to the low amount of these cells found in
various tumor tissues. Further studies on melanoma (17); neuroectodermal tumor (18); lung (19), ovarian (20), and colon cancer (21); and
recently on breast cancer types (22–24) have shown that TIL-B cells
are of potential interest. Immunocompetent B cells in some solid tumors were contributed to spontaneous tumor regression as well (25).
Both naturally occurring and vaccine-induced Ab responses to some
breast cancer Ags could be associated with improved survival in some
cases (26).
We postulate that TIL-B cells have specific tumor-recognizing
capacity, and could serve as a new source besides peripheral blood
and lymph nodes to search for tumor-binder Abs. We postulate that
the analysis of the Ab repertoire of these TIL-B cells could lead to
the further understanding of the precise nature of natural Abs to
tumor Ags.
3
Abbreviations used in this paper: TIL-B, tumor-infiltrating B; AP, alkaline phosphatase; dAb, Ab derivate; IPTG, isopropyl ␤-D-thiogalactoside; MBC, medullary
breast carcinoma; R, replacement; S, silent; scFv, single-chain variable fragment.
0022-1767/05/$02.00
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The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer
biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration
paralleled to a relatively better prognosis. Ig-rearranged V region VH-JH, V␬-J␬, and V␭-J␭ genes, amplified by RT-PCR of the
infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable
fragment Ab minilibrary was constructed out of randomly selected VH and V␬ clones and tested for binding activity. Our data
analysis revealed that some of the VH-JH, V␬-J␬, and V␭-J␭ region sequences were being assigned to clusters with oligoclonal
predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected
out of the single-chain variable fragment ␬ minilibrary tested against membrane fractions of primary breast tumor cells and
tumor cell lines, the VH of which proved to be the overexpressed VH3-1 cluster. The specific binding was confirmed by FACS
analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells
infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell
progression and could be used for further development of diagnostic and/or therapeutic purposes. The Journal of Immunology,
2005, 175: 2278 –2285.
The Journal of Immunology
To address this hypothesis, a detailed Ig repertoire analysis of
the expressed human medullary breast carcinoma (MBC) TIL-B
cell Ig V regions was performed. Our model system for characterizing the expressed Ig repertoire (22, 27, 28) was established
from high grade MBC because this has the highest B cell infiltration (29). A combinatorial single chain Fv ␬ (scFv␬) minilibrary
from selected VH and V␬ region genes was generated and tested
thereafter for breast tumor cell-binding capacity. The findings presented in this work provide a new strategy for identifying novel
tumor-specific Ags, and serve as proof of principle for the potential
tumor Ag-binding capacity of B lymphocytes infiltrating human
breast carcinomas.
Materials and Methods
PCR amplification of TIL-B Ig V regions
Cloning and sequencing of VH-JH, V␬-J␬, and V␭-J␭ TIL-B Ig
regions
PCR products were purified and blunt end ligated into pUC18 (SmaI/BAP)
plasmid vector (Pharmacia Biotech). Escherichia coli TG1 bacteria were
transformed, and positive clones were selected by PCR, as described (22,
28). Sequencing of plasmid dsDNA (QIAprep Spin Miniprep kit; Qiagen)
was performed partly by Sequence Version 2.0 DNA Sequencing kit (USB)
and mainly by automatic sequencing (Dye Terminator Sequence Reaction
Kit, DyeEx Spin kit (Qiagen; ABI PRISM Software, automatic sequencer
of PerkinElmer). More than 60 VH, V␬, and V␭ clones were sequenced,
respectively. Further comparative DNA sequence analysis was performed
by accessible software and databases available through the Internet: BIOEDIT 5.0.9 (31) was used for editing sequences, Clustal X 1.8 (32) for
sequence alignment, and TREEVIEW 1.5.2 (33) for displaying sequence
trees based on homology level. Sequence comparison was made to KABAT National Institutes of Health (具http://immuno.bme.nwu.edu典), IMGT
(具http://imgt.cines.fr.典), GenBank, Embnet via National Center for Biotechnology Information Blast Engine (具www.ncbi.nlm.nih.gov/BLAST/典), and
SRS (具http://srs.hgmp.mrc.ac.uk典). Our sequences were compared with
germline sequences and related coding regions (V, (D), J) participating in
the whole length of the V region according to the International ImMunoGeneTics database results (34) and referred to compiled germline. To find
homologous sequences, a databank search via National Center for Biotechnology Information Blast server to GenBank/European Molecular Biology
Laboratory Net databases was conducted and the generated data termed as
Blastn result. More details about data analysis will be published elsewere
(35).
Construction of combinatorial scFv␬ minilibrary
The construction of the scFv␬ minilibrary was conducted, as described (27,
28), by selecting VH and V␬ genes and amplified with specific primers
based upon DNA sequence analysis. Assembly reactions of the selected
rearranged Ig V region H (26VH) and L chain (32V␬) genes were conducted by a three-step PCR amplification, using a linker peptide (Gly4Ser3)
coding sequence. The purified and SfiI- and NotI-digested V␬-J␬ fragments
were ligated into pHEN1 phage vector, and E. coli TG1 bacteria (Stratagene) were infected according to methods (36) slightly modified. The
scFv␬ combinatorial minilibrary generated had 832 possible combinations
and 5 ⫻ 106 members in size.
PCR screen and fingerprint analysis of scFv inserts
The scFv␬ combinatorial minilibrary was plated on selective culture medium (100 ␮g/ml ampicillin 1% glucose 2⫻TY) at 30°C. Ninety clones
were randomly picked, and PCR screening for the scFv insert was performed using LMB2 (forward) and LMB3 (backward) primers and standard conditions (30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for
1.5 min, followed by an extension at 72°C for 10 min). A masterplate was
set up in 2⫻TY ampicillin/glucose culture medium containing 15% glycerol and kept frozen until further usage. In addition, the amplified PCR
samples were digested with 5 U of the enzyme HaeIII for 1 h at 37°C, and
the restriction pattern of fingerprint analysis was visualized by gel electrophoresis on 3% agarose.
Cell membrane preparation of fresh cultured tumor cells and
cell lines
Cell lines of different histological origin (MDA-MB 231 and MDAZR75-1 breast cancer cell lines, 293 kidney epithelial cell line, LS174T
colorectal carcinoma line, SK MEL-28 melanoma cell line) and the control
COS7 cell lines were obtained from American Type Culture Collection.
They were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% (v/v) heat-inactivated FCS (Invitrogen Life Technologies), 50 ␮g/ml penicillin (Invitrogen Life Technologies), 100 ␮g/ml streptomycin, and 2 mM L-glutamine (Invitrogen Life Technologies). All cells
were cultured at 37°C in a 5% CO2 atmosphere. Growth medium was
changed every 3 days, and cells were subcultured according to their growth
rate. Small pieces of tumor tissue (0.6 ⫻ 0.6 ⫻ 0.6 cm), aseptically obtained after surgery from two patients with breast carcinomas, were minced
under medium supplemented with 20% culture supernatant from the 293
cell line in growing phase and 0.01% sodium pyruvate and kept under
suitable conditions. Cultured cells (1 ⫻ 107 each) were processed further to
obtain membrane fractions, as described (37).
Soluble scFv ELISA on membrane preparation
Ninety-six-well Maxisorp (Nunc) microtiter plates were coated (16 h, 4°C)
with 1–10 ␮g of membrane preparations of breast carcinoma cells and
control cell lines. Plates were washed five times with PBS and blocked with
200 ␮l of 2% BSA (Sigma-Aldrich) in PBS. Soluble scFv production was
induced from individual bacterial clones by standard isopropyl ␤-D-thiogalactoside (IPTG) induction procedure. In ELISA, when testing soluble
scFv fractions against tumor cell membrane preparations, anti-c-myc mAb
(Sigma-Aldrich), HRP-conjugated anti-mouse Ab (Amersham), and the
ABTS (Boehringer Mannheim) were used and analyzed by an ELISA
reader (415 nm) (MWG Biotec). In further blocking experiments, selected
hybridoma supernatants with known specificity, alkaline phosphatase
(AP)-conjugated anti-c-myc Ab (Sigma-Aldrich), and p-nitrophenyl phosphate (Sigma-Aldrich) subtrate system were used, and the reaction was
evaluated at 405 nm.
Dot-blot analysis of gangliosides
A total of 5 ␮g of seven different gangliosides (GD1a, GD1b, GD2, GD3,
GM1, GM2, and GM3; Calbiochem) was placed on silica plates (2–20 ␮m
porus size; Sigma-Aldrich), dried, and blocked with PBS/2% milk for 2 h.
Soluble fraction (10 ␮l) produced by the tumor-binder TIL-B scFv␬ bacterial clone was added onto the dots containing the targeted gangliosides
and dried. After washing and drying the plates, AP-conjugated anti-c-myc
Ab in PBS-BSA (1%) and bromochloroindolyl phosphate/NBT substrate
(Sigma-Aldrich) were used for analyzing the binding capacity. Negative
controls were set up with culture supernatants of pHEN1 vector/no insert
containing E. coli TG1 bacteria. As a positive control, we used supernatant
from the hybridoma, HCB-C3 with known GD3 specificity (M. Glassy,
unpublished observations). To evaluate the binding potential of the G2scFv
Ab derivate (dAb) fragment to purified gangliosides and different relevant
cell lines, further dot-blot experiments were set up with membrane preparations of breast tumor cells (TU1, TU2, MDA-MB231) and irrelevant
cell lines (COS7).
FACS analysis with soluble scFv fractions
From two patients with invasive ductal carcinoma, two separate breast
cancer cell lines were established from the surgically removed tissues.
Cells from log-phase growing cultures with ⬎95% viability were gently
scraped off, and 3 ⫻ 105 cells per test tube were used. Soluble scFv fraction
of the binder-positive clone was prepared by standard IPTG induction
method in large scale. Tumor cells were incubated with soluble scFv Ig
fraction (diluted 1/1 in FCS RPMI 1640) at 37°C for 1 h in pretreated (1%
BSA) plastic tubes. After three washes with FCS RPMI 1640, anti-c-myc
9E10 mAb (Sigma-Aldrich) was added for another 1-h incubation. Three
washes with 1% BSA PBS were followed by administration of FITC antimouse IgG F(ab⬘)2 (Sigma-Aldrich) for 1 h at 4°C. Cells were washed three
times with 1% BSA PBS and PBS, and fixed in 1% Formalin-PBS. Ten
thousand cells were counted in a FACSCalibur (BD Biosciences) and analyzed by CellQuest.
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Tumor tissue was obtained aseptically during surgery from a patient with
MBC. The tissue sample was minimized for attached normal tissue. RNA
was extracted according to the manufacturer’s instructions (RNeasy Mini
kit; Qiagen), while cDNA was synthetized by the commercially available
kit (Pharmacia Biotech). To amplify the human Ig VH-JH, V␬-J␬, and
V␭-J␭ encoding regions, specific primers were designed (30) and PCR was
performed (35 cycles: 1 min, 94°C; 1 min, 60°C; and 1 min, 72°C;
PerkinElmer/Cetus thermocycler).
2279
2280
NOVEL GANGLIOSIDE IDENTIFIED BY B CELLS IN BREAST CARCINOMAS
Results
Revealing the V gene usage, mutational pattern, and other
characteristics of B cells infiltrating MBC by comparative DNA
sequence analysis
FIGURE 1. Immunohistological staining of a paraffin-embedded tissue
section of lymphocytic MBC. H&E staining. Magnification, ⫻270. Arrow
shows massive lymphocyte infiltration, the great part of which are B lymphocytes and plasmocytes.
FIGURE 2. Cut pie chart shows all analyzed sequences (111) distributed to families and clusters. Different families are with different colors,
while distinct clusters make distinct slices. The number at a given slice
indicates the number of members in the cluster. The most relevant clusters
are pulled out for better view. We have chosen the VH3/1, VH5, V␬1/1, and
V␭3/1 for closer examination.
differed ⬎5% to that one (Fig. 3C). Representative data about the
V␭3 L chain family showed an overrepresented cluster (V␭ 3/1)
with 90% of the sequences with the same germline origin (data not
shown).
In the case of selected representative VH and VL families, we
investigated the number of the members belonging to one defined
cluster, the length of sequences, and the number of replacement
and silent (R/S) mutations in the framework, as well as in the
CDRs. The length of the CDR3 region; the deletion and/or insertion mutations in the V, D, and J regions; and the percentage of
identity in CDR3 were also analyzed. The VH, V␬, and V␭ lengths
defined were 351 and 369 bp in the overrepresented VH3 and VH5
families, 324 and 342 bp in the representative V␬ families, and 333
and 321 bp in the selected V␭ cases. A high number of R as well
as S mutations was found in the framework regions of the overexpressed VH3/1 cluster that was not observed in the VH5 family
(Table I). A relatively high R/S ratio in CDR3 was characteristic
only for the VH3/1 cluster. Deletion mutations caused the low homology level of CDR3, in comparison with the germline region. A
very high internal homology level and a low closest germline homology were characteristic of the VH3/1 cluster. The V␬4/1 cluster
showed a 3.3 R/S mutation ratio, whereas mutations in the V␭
families were difficult to detect. CDR1, CDR2, and CDR3 region
sequences of the most relevant clusters were compared with each
other and the blast query result. DNA mismatches were defined to
each other and to the germline sequences, and the gaps representative for the given CDR3 were determined as well (Table I).
Characterization of the scFv␬ combinatorial minilibrary and
selection of tumor-binder clone
Ninety-seven percent of the clones of our scFv␬ library (5 ⫻ 106
members) contained an insert. Of these, 45% contained a scFv
insert (⬃800 bp), and 54% of clones had a 600-bp truncated scFv
(Fig. 4). Thirty-three percent of the clones with an insert of 600 bp
showed identical fingerprint patterns (data not shown). DNA sequencing showed that these truncated clones were comprised of a
VH, the linker, and a part of the framework region 1 of V␬.
Of 80 soluble scFv-producing bacterial clones (exhibiting either
the 800- or the 600-bp insert) tested by ELISA, 1 clone (G2/clone
15) showed a binding capacity to membrane fractions of fresh
cultivated breast cancer cells of an invasive breast carcinoma patient, and of the MDA MB-231 breast cancer cell line (Fig. 5). No
binding could be detected to the LS174T colorectal cancer, 293
kidney epithelial, and the control COS cell lines. The result shows
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A significant lymphocyte cell infiltration was found in human
MBC, as shown in Fig. 1.
VH-JH, V␬-J␬, and V␭-J␭ V regions were cloned, and the V
gene usage was analyzed. Of the clones with the expected insert
size, 67 VH (460 bp), 72 V␬ (440 bp), and 63 V␭ (430 bp) clones
were selected for sequencing. The VH, V␬, and V␭ sequences
could be grouped into different families based on searching for the
original germline sequences by IMGT database. Expressed Ig V
regions were determined using the KABAT database, and characteristics were defined using commercially available softwares. Fifty-one percent (24 of 47) of the VH region sequences belong to the
VH3 family, and the others are members of the VH4 (23.4%), VH5
(21.2%), and VH1 (4.3%) families. We found the V␬1 as being the
most represented family (19 of 38 (50.0%)), followed by V␬4
(29.0%), V␬2 (18.4%), and V␬3 (2.6%). Based on the highest
homology level (90 –100%) to IMGT-defined germline sequences,
a family ranking has been made. TIL-B VH and VL sequences with
the same compiled germline sequence were grouped into one cluster. In most of the families, clusters with high internal homology
(94.5–99.8%) could be distinguished. Some clusters defined were
overrepresented, while others contained just a few members (Fig.
2). The VH3 family sequences (72%) could be grouped into the
VH3/1 (10 of 22 (45%)) and VH3/4 (5 of 22 (23%)) main clusters.
Of the eight clusters, 42% (8 of 19) of the V␬1 sequences belong
to the overrepresented one. The V␭ V region sequences showed
less variability as the three families were represented by approximately equal members, that is 26.9% (7 of 26) V␭1, 38.5% (10 of
26) V␭2, and 34.6% (9 of 26) V␭3.
Sequences belonging to the defined clusters were grouped into a
tree structure. The overrepresented VH3/1 cluster showed a high
difference (⬎12%) in comparison with all other VH3 sequences.
While compiling the nearest germline sequences to our defined
clusters, some genes seem to participate in more cases. Some combinations are more frequent (VH3a/D4a/J4a) than others, suggesting more efficiency in the representation. By contrast, when the
same VH segments were combined with the above different D or J
segments (VH3a/D4b/J4b), they resulted in underrepresentation of
the given Ig. The tendency in how the nearest VH3 sequences build
groups is depicted in Fig. 3A. Contrary to that, the divergence
among sequences of VH5 family is very low (0.2– 0.4%), and these
sequences share the closest germline sequence found (Fig. 3B).
Divergences among different V␬1 sequences may be significant.
Based on a very low divergence level (0.2%) and one mutual
germline sequence, eight V␬ sequences could be classified into the
overrepresented cluster (V␬1/1). All of the other V␬1 sequences
The Journal of Immunology
2281
that by this strategy already, without previous panning procedures,
just after setting up a master plate from the scFv␬ library in one
96-well ELISA plate, we were able to obtain a tumor-binder clone.
After sequencing the positive clone (G2/clone 15) and making
comparative data analysis with BLAST, a 98% identity in the VH
region to a GD2/GD3-binder Ab was found. CDR1, CDR2, and
CDR3 VH sequences showed high identity level between the G2
clone and that of the GD2/GD3-binder VH sequence (Fig. 6). The
truncated V␬ L chain belongs to the V␬4 family. There was one
nucleotide difference in the linker sequence of this scFv dAb G2
clone. Interestingly, the G2 clone had only expressed a 25-nt-long
region of a V␬ chain. Sequence analysis of nine randomly picked
negative clones from our masterplate showed that three of them
had a full-length DNA as scFv, and two others were truncated
scFv-s (with a truncated VH in framework region 1 at the 5⬘ end).
The four other clones were of smaller size (truncated scFv-s), with
complete (3) or shortened (1) VH regions fused either to a complete (3 case) or shortened (1 case) linker sequence and a
truncated V␬.
before the soluble scFv dAb G2. The direct binding of soluble scFv
dAb G2, detected using an AP-labeled anti-c-myc Ab, was inhibited by the anti-GD3 mAb HCB-C3 (up to 75%) (Fig. 7). When
another ganglioside-specific Ab (HCB-C3) and indifferent control
Ig were added, there was no blocking effect. In addition, the scFv
dAb G2 showed reactivity to a dried cell membrane ghost preparation of the GD3 ganglioside-positive SK MEL-28 (human melanoma cell line), whereas there was no binding to LS174 colon
carcinoma cell ghost, known to be ganglioside negative. Finally,
the soluble fraction of scFv dAb G2 bound to GD3 ganglioside on
silica gel-coated membrane in blotting experiments. There was
only a marginal or no staining to the other six ganglioside types or
the negative control sample. Binding capacity of our G2 scFv dAb
fragment to gangliosides and membrane preparations of some relevant breast cancer and other cell lines loaded on TLC plates
showed evidence of GD3 specificity (Fig. 8).
Characterization of the binder TIL-B scFv clone shows evidence
of GD3 ganglioside binding
Two adherent robust cell lines, TU1 and TU2, were established out
of the separated and cultured cancerous tissues of two patients with
invasive ductal breast cancer (Fig. 9). Although a detailed characterization of these lines will be published elsewhere, some features
concerning ganglioside expression are explained and presented in
Fig. 10. The reaction pattern with HCB-C3 ganglioside D3-specific Ab and culture supernatants containing soluble scFv antiGD3 (G2 clone, truncated scFv, B2 clone, whole scFv) fragments
ELISA-blocking experiments were performed with human or
mouse hybridoma supernatants having known ganglioside-binding
capacity. Membrane fractions of the MDA MB-231 breast carcinoma cell line and of fresh cultivated tumor cells were coated to
polystyrene plate and incubated with soluble scFv dAb G2 clone.
In other experiments, the GD3-reactive Ab (HCB-C3) was added
Soluble scFv Ab fragments out of the TIL-B minilibrary show
binding capacity to invasive breast cancer cells by ELISA and
FACS analysis
Table I. DNA analysis of the most abundant TIL-B Ig VH and VL clustersa
Identity %
Cluster
(members no.)
VH3/1 (11)
VH5 (10)
V␬1/1 (8)
V␬4/1 (6)
V␭2 (10)
V␭3/1 (9)
Length
R/S in FR
Mutation
R/S in CDR 1, 2
Mutation
351
369
324
342
333
321
18/11
1.9/0
0.4/0
3.3/0.15
1/0.1
0.1/1
6/1
1.0/0
0/0
1.7/0
0.4/0
0/0
CDR3 Length/Deletion (d)
Insertion (i)/Identity %
31/Jd2/65
47/Dd7/72
22/Vd2/74
23/0/86
35/Vd4,Jd2/83
30/Jd4/80
Internal
Germline
Blastn
99.5
99.4
98.8
94.5
99.0
97.8
80.5
94.9
97.6
96.2
97.6
99.9
98.1
90.0
96.7
95.3
98.2
99.3
a
Total lengths of the cloned sequences, ratio of R/S mutations in framework regions (FR) 1, 2, 3, and 4, CDR1 and CDR2 regions, and the average mutations per sequence
are indicated. The CDR3 length includes the R/S region gaps. The percent of identity refers to the germline sequence (IMGT database was used).
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FIGURE 3. A, The tree of the TIL-B Ig VH3 family includes all of the VH3 family sequences analyzed. Two main groups might be distinguished: the
first consists of one cluster (VH3/1), while the other is rather heterogenous, and shows ⬎10% divergence from the first group. The compiled nearest germline
region sequences are depicted as: a, VH33a-D4a-J4a (that is the selected VH3/1); b, VH3b-D4-J6; c, VH3a-D4b-J4b; d, VH3f-D6-J4; e, VH3c-D4-J4; f,
VH3c-D1-J4; g, VH3e-D3-J4; h, VH3d-D3-J3. Small boxes in the tree structure indicate the sequences. The number in small boxes represents the number
of sequences grouped together; no number represents one sequence. B, The tree of TIL-B Ig VH5 family, which is characteristic for its internal homology.
Only one or two sequence mismatches are found between the members of the family. C, The tree of TIL-B Ig V␬1 family includes all sequences. The
sequences divide into two groups. One is with close identity separated to the two sides of the tree. The other in the middle of the tree is rather heterogenous.
The compiled nearest germline region sequences are depicted as: a, V␬1a-J1; b, V␬1b-J1; c, V␬1c-J1; d, V␬1d-J1; e, V␬1e-J4; f, V␬1d-J3; g, V␬1a-J2.
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NOVEL GANGLIOSIDE IDENTIFIED BY B CELLS IN BREAST CARCINOMAS
FIGURE 4. PCR screening of the scFv␬ combinatorial minilibrary originating from VH and V␬ regions of B lymphocytes infiltrating a human
MBC. The light lanes with 800 and 600 bp of size represent positive clones
for insert of full-length scFv␬ or truncated scFv␬, respectively. St, Represents a standard marker (Gene Ruler 50-bp DNA ladder; Molecular Biology, Inc. Fermentas), whereas samples 1–19 are the randomly selected 19
clones.
Discussion
The nature of the human Ig repertoire in cancer patients is of interest and could provide important insight into aspects of tumor
biology. The TIL-B cells that accumulate in solid tumor tissues
could be a potential source for identifying novel tumor Ags. The
data presented in this work on comparing TIL-B cell Ig V region
sequences at the DNA level confirm our earlier observations on the
oligoclonal nature of Ig repertoire in MBC (22) and suggest that
the TIL-B Ig repertoire may be Ag driven. In the defined four VH,
four V␬, and three V␭ families, some overrepresented clusters
could be distinguished according to the tree analysis. The VH5 and
selected V␭ families showed internal similarity, whereas VH3 and
V␬1 families were moderately diversified. Some characteristic features of early B cells have been observed, such as a rather low
mutation rate distributed in Ig framework regions and a significant
internal sequence homology. Interestingly, a higher than 3 R/S
ratio in CDR regions was found only in the selected overrepre-
sented clusters. Our earlier and present results together with the
recently published data about ductal breast carcinoma and MBC
(22, 24, 35, 38) suggest that at least part of the B cell repertoire
of TIL-B cells is tumor-associated Ag driven (39 – 41). The focus
of our study was to obtain experimental proof of this Ag-driven
response by identifying TIL-B-specific tumor-binder cancerrestricted clone(s).
Our investigations suggest that a tumor-restricted response of
clonally selected TIL-B cells in MBC does exist. We selected a
positive tumor-binder scFv (dAb VH) clone from our scFv␬ minilibrary that showed strong restricted binding to invasive ductal
breast carcinoma cells. Although this positive clone was first
thought to be a truncated scFv with only a functional VH domain,
it showed a tumor-restricted pattern and significant binding potential to invasive ductal breast carcinoma cells. Such VH-binding
ability has been previously detected in melanoma (42) and autoimmune cases (43). DNA sequence analysis showed that our VH is
a member of the overrepresented VH3/1 cluster. The VH3/1 cluster
showed very high homology to the VH of a GD2/GD3 gangliosidebinder Ab. In addition, one isolated clone from the V␬4/1 cluster
showed a high identity to a ganglioside (GM3)-binder Ig VL chain
(data not shown).
Gangliosides have been extensively investigated for their structural and functional properties (44, 45), as well as their role in
tumor cell transformation and potential capacity as suitable tumor
targets for diagnostics or therapeutics (46, 47). In addition to the
well-defined GD2 overexpression in neuroblastomas and melanomas (48, 49), GD3 may be a useful marker in breast carcinomas.
As GD3 gangliosides are poorly immunogenic, it has been difficult
to obtain human Abs to them. Therefore, a new source and method
of obtaining Ab fragments that react with the ganglioside D3 on
breast cancer may be significant. Because GD3 is associated with
invasive ductal carcinomas (50, 51), it is of interest to discover
FIGURE 6. Ig VH CDR1, CDR2, and CDR3 protein sequences in oneletter code of the positive clone compared with the database search result.
Dot, amino acids missing; dashed lines, identity.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
on ganglioside-expressing cells showed further evidence concerning GD3 binding of the selected clones. Mid-log phase cells
showed strong HLA Ag positivity with the W6/32 mAb, and
MUC-1 positivity of breast tumor TU2 cells was found (Fig. 10).
The surface-attached cultured breast cancer and melanoma cell
lines suddenly detached from the chamber slide after the reaction
with anti-ganglioside Abs, giving a further indirect evidence about
the targeted structures, as that is a characteristic feature of gangliosides. In the immunofluorescence experiments, the mouse
mAb 9410 to anti-c-myc was used as a control, and it was negative.
Culture supernatants containing soluble scFv anti-GD3 (G2 clone,
truncated scFv) showed positive binding to both the TU1 and TU2
breast cancer cell lines. As the binding intensity was similarly
strong, only data with the TU2 invasive ductal breast cancer cells
are shown (Fig. 11A). Other selected clone supernatants from the
phage library with the same overexpressed VH3/1 VH DNA sequence, but representing a whole scFv fragment (B2 scFv), also
gave positive fluorescence with the given cells (Fig. 11B). Similarly, incubation with soluble scFv fragments, obtained after panning against the MDA-MB 231 cell line, resulted in significant
positive tumor cell binding (Fig. 11C). The SK Mel-28 human
melanoma cell line, being strongly positive for the ganglioside
GD3, was the target for the hybridoma HCB-C3. The immunofluorescence labeling reaction with HCB-C3 anti-GD3 Ab against the
TU1 and TU2 breast cell lines was significant. However, binding
of the G2 scFv dAb and the immunofluorescence positivity against
breast tumor cells was even stronger, while no remarkable labeling
to normal cells was found (B. Kotlan, J. Toth, J.-L. Teillaud, M.
McKnight, and M. Glassy, manuscript in preparation).
FIGURE 5. Soluble scFv ELISA. Soluble scFv-containing supernatants
of IPTG-induced bacterial clones were incubated with membrane fractions
of fresh short-time cultivated breast tumor cells and different tumor cell
lines (MDA MB-231, LS174T, COS7). An anti-MUC scFv was used as
positive control, and a supernatant of pHEN1 (no insert) TG1 clones as a
negative control. ELISA OD data (␭: 415 nm) of one representative experiment (of four repeated experiments) are presented. G2 represents the
positive binder clone.
The Journal of Immunology
whether our TIL-B-derived Ab fragments recognize this Ag. Because no anti-GD3 Ab fragments from TIL-B cells have been reported to date, our data serve as first proof and potential for obtaining these human Abs out of MBCs. The importance and
capacity of gangliosides and ganglioside-specific Abs as markers
of tumor cells have been investigated (52–55), and therapeutic
trials have been put on the way (56 –58).
These results establish a theoretical and experimental basis of
our original hypothesis about the targeted tumor cell-binding capacity of the overexpressed Ig V regions of TIL-B cells. A direct
selection of overrepresented clusters based on Ig repertoire anal-
FIGURE 8. Dot-blot experiment on silica gel-coated membranes with
gangliosides and ganglioside-expressing cell lines shows the binding capacity of G2-soluble scFv dAb to GD3 ganglioside (S4), and gangliosidepositive invasive ductal breast carcinoma cell lines TU1 (S5), TU2 (S6),
MDA-MB231 (S7), but no binding to indifferent COS7 cell line (S1), other
gangliosides such as GD1 (S2), GM2 (S3), or the background diluting
solvent (S8). AP-conjugated anti-c-myc Ab and bromochloroindolyl phosphate/NBT substrate were used for reaction development.
FIGURE 9. Photo of the TU2 breast cancer cell line in culture, which
was established from invasive ductal breast carcinoma tumor tissue in our
laboratory.
ysis for building a scFv minilibrary can simplify and shorten the
process for searching for tumor target and tumor-reactive binder
Abs. Our results provide a potential new strategy for identifying
novel tumor Ags and can serve as a proof of principle for demonstrating the potential tumor Ag-binding capacity of B lymphocytes infiltrating human breast carcinomas and other solid tumors.
Furthermore, our results suggest that it may be worthwhile to investigate other types of solid tumors with heavy or moderate lymphocytic infiltration.
In addition, the identified tumor Ags might be used in the development of new tumor diagnostic and immunotherapeutic drug
discovery programs (59 – 61). TIL-B lymphocytes could provide a
new source for the production of novel Ab fragments with improved properties for applications such as tumor targeting in vivo.
In well-differentiated ductal carcinoma in situ and invasive ductal
carcinomas (grade 1), numerous cells exhibited cell surface labeling, which was reported to be absent in moderately and poorly
differentiated tumors of the same types. The fact that in malignant
lesions an abnormal distribution pattern of O-acetylated disialogangliosides (GD3) might be defined in comparison with a benign
proliferation renders the ganglioside a putative prognostic and diagnostic marker (62). Our GD3-specific Ab fragment labels the
more aggressive MDA-MB 231 cells that lack the estrogen receptors and are not responsive to estrogen and anti-estrogens (tamoxifen and benzothiophene). Ganglioside expression varies significantly among different cell lines, and both GM3 and GD3 are
suggested to be involved in regulation of growth factor functions
and tumor cell proliferation (51). Because gangliosides are overexpressed and cancer-restricted molecules, they are promising targets in the field of cancer research (52, 56, 59, 60, 63).
FIGURE 10. Compared characteristics of novel breast cancer cell lines
by ELISA. The reaction pattern of breast cancer TU2, MDA MB231,
MCF-7, and MEL-SK28 membrane fractions with ganglioside-specific Abs
and our G2 scFv dAb fragment. Anti-c-myc Ab and AP-labeled anti-mouse
second Ab with p-nitrophenyl phosphate substrate were used in the ELISA
experiment. In 1, HCB-C3 anti-GD3; 2, G2scFv; 3, B2scFv; 4, W6/32; and
5, anti-MUC-1 Abs were used. In 6 and 7, controls with pHEN1/TG1
culture medium and PBS/BSA, respectively.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. ELISA blocking. Soluble scFv containing supernatant of
the binder G2 clone given alone (f) or together with an anti-GD3-specific
Ab (䡺) to membrane fractions of fresh short-time cultivated breast tumor
cells (TU2) (A1, B1) and the MDA MB-231 cell line (A2, B2). When Abs
with other ganglioside specificities (HCBD1) and control Ig preparations
were given together with the G2 clone-soluble scFv dAb G2 clone (s),
there was no change in binding efficiency (C1, C2, D1, D2). Mean OD
values (␭: 405 nm) of one representative experiment (of three experiments)
using AP-conjugated anti-c-myc Ab are shown. Mean control samples of
pHEN1/TG1 supernatants (E1,2) and PBS/BSA (F1,2) are presented as
dotted bars.
2283
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NOVEL GANGLIOSIDE IDENTIFIED BY B CELLS IN BREAST CARCINOMAS
FIGURE 11. FACS analysis. Fluorescence intensity histograms show binding of soluble scFv Ab fragment scFv aAb (G2) (A), scFv (B2) (B) clones,
and scFv of whole library after panning against MDA MB 231 (MDApscFv) (C) to TU2 invasive ductal breast cancer cells. Negative background control
data with anti-c-myc mAb and FITC-labeled anti-mouse IgG are presented as gray histograms.
19.
20.
21.
22.
23.
24.
Disclosures
The authors have no financial conflict of interest.
25.
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