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Plenary paper
Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen
Tzu-Hsuan Huang, Pin-Yi Wu, Chin-Nien Lee, Hsing-I Huang, Shie-Liang Hsieh, John Kung, and Mi-Hua Tao
The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumorspecific antigen but is only weakly
immunogenic. This study demonstrates
that the immunogenicity of the tumor Id
protein can be dramatically increased by
directing it to antigen-presenting cells
(APCs). Cytotoxic T-lymphocyte antigen
4 (CTLA-4) present on activated T cells
has a strong binding affinity to both B7-1
and B7-2 molecules, which are primarily
expressed on APCs. After construction of
a fusion protein consisting of Id and
CTLA-4 (Id-CTLA4), mice immunized with
the fusion protein induced high titers of
Id-specific antibody and T-cell proliferative responses without adjuvants and
were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was
so potent that even low doses (down to
0.1 ␮g) of the immunogen were able to
elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the abil-
ity to bind B7 molecules on APCs was
shown to be required for the enhanced
immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a
potential tumor antigen represents an
effective approach to prime antitumor
immunities in vivo and may be applicable
to the design of vaccines for a variety
of other diseases. (Blood. 2000;96:
3663-3670)
© 2000 by The American Society of Hematology
Introduction
The idiotypic protein (Id) of the surface immunoglobulin (Ig)
expressed by malignant B lymphocytes can serve as a unique
tumor-specific antigen (Ag) and as a model for the development of
cancer vaccines. Vaccination with tumor-derived Id induces protective immunity against lymphoma in a number of mouse models.1-6
Moreover, in combination with chemotherapy, Id vaccination was
reported to cure animals with established tumors.7,8 In a clinical
trial for low-grade follicular lymphoma, Id immunization induced
humoral responses against the specific Id expressed on each
patient’s tumor.9 However, the autologous Id proteins, like many
other tumor Ags, are only weakly immunogenic and must be
chemically coupled to a strongly immunogenic carrier protein and
mixed with an adjuvant to induce an immune response. Alternatively, genetic fusion of the Id protein with various cytokine
molecules, including interleukin (IL)-2, IL-4, and granulocytemacrophage colony-stimulating factor (GM-CSF), converts this
self tumor Ag into a strong immunogen capable of inducing anti-Id
antibody (Ab) and protective immunities without carrier proteins or
adjuvants. This adjuvant effect is dependent on the biologic activity
of the cytokine as well as the covalent linkage of the Id and
cytokine.10,11 Vaccination with fusion proteins between a singlechain Id and chemokine was also recently reported to induce
protective immune responses against a large tumor challenge.12
Another strategy to increase the immunogenicity of a protein
Ag is by directing immunogens to antigen-presenting cells (APCs)
for more efficient Ag processing and presentation. Previous
experiments have shown that coupling immunogens to Abs against
the class II major histocompatibility complex (MHC), the Fc␥
receptor (Fc␥R), the 33D1 dendritic cell-specific Ag on APCs,13,14
or surface IgM or IgG on B cells15 can substantially increase
immune responses. Boyle and colleagues16 showed that vaccination with DNA encoding a model Ag (human IgG) and cytotoxic
T-lymphocyte antigen 4 (CTLA-4) dramatically increased both Ab
and T-cell proliferation responses compared with the control
plasmid expressing the Ag alone. CTLA-4 is a glycoprotein
expressed on activated T cells that has a strong binding affinity to
both B7-1 (CD80) and B7-2 (CD86) molecules, which are primarily expressed on APCs.17 Presumably, the direct targeting of the Ag
to APCs through the interaction of CTLA-4 and B7 causes the Ag
to be processed and presented to T cells with much higher
efficiency, therefore leading to a stronger immune response.
In the present study, we investigated whether the immunogenicity of
the tumor Id protein could be improved by targeting to APCs through
B7-1 and B7-2 molecules. We constructed a fusion protein consisting of
38C13 Id and CTLA-4 and found that the resulting Id-CTLA4 fusion
protein induces significant antitumor immunity. The Id-CTLA4 fusion
protein was so potent that even a dose as low as 0.1 ␮g was able to elicit
high titers of anti-Id Abs. These results demonstrate that fusion of a
weak tumor Ag to CTLA-4 represents a powerful means to increase the
immunogenicity of the Ag and may be applicable to the design of
vaccines for immunotherapy of other types of tumors as well as for other
pathogens and disease states.
From the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;
Graduate Institute of Medical Technology, National Taiwan University, Taipei,
Taiwan; Department of Microbiology and Immunology, National Yang-Ming
University, Taipei, Taiwan; and Institute of Molecular Biology, Academia Sinica,
Taipei, Taiwan.
T.H.H. and P.Y.W. contributed equally to this paper.
Submitted February 16, 2000; accepted August 3, 2000.
Supported by Academia Sinica and by grant DOH89A1-PPLABAD01 from the
National Health Research Institute, Taiwan.
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
Materials and methods
Mice
Female C3H/HeN mice, 6 to 8 weeks old, were purchased from the National
Laboratory Animal Breeding and Research Center, Taipei, Taiwan, and
Reprints: Mi-Hua Tao, Institute of Biomedical Sciences, Academia Sinica,
Taipei 115, Taiwan; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
3663
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3664
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
HUANG et al
housed at the Laboratory Animal Facility, Institute of Biomedical Sciences,
Academia Sinica, Taipei, Taiwan. All animal studies were approved by the
Animal Committee of Institute of Biomedical Sciences, Academia Sinica
and performed according to their guidelines.
Cell lines
The carcinogen-induced 38C13 B-cell lymphoma has been described18
previously and was a gift from R. Levy (Stanford University, Stanford,
CA). Transfectoma cell lines producing a human-mouse chimeric Id or
Id-murine GM-CSF fusion protein (Id-GM) were described previously.10
FO (CRL-1646, American Type Culture Collection, Manassas, VA) is a
murine plasmacytoma cell line negative for Ig production. DC2.4 (courtesy
of K. Rock, Dana Farber Cancer Institute, Boston, MA) is an immortalized
murine bone marrow–derived dendritic cell line and is positive in the
expression of B7-1 and B7-2.19 DG44 (courtesy of L. Chasin, Columbia
University, New York, NY) is a dihydrofolate reductase–deficient mutant
cell line of Chinese hamster ovary (CHO) cells.20
Plasmid construction
The complementary DNAs (cDNA) of murine B7-1 (CD80) and B7-2
(CD86) were kindly provided by G. Freeman (Dana-Farber Cancer
Institute) and separately cloned into a eukaryotic expression vector,
pTCAE,21 under the transcriptional control of a cytomegalovirus promoter.
The resulting plasmids containing B7-1 or B7-2 genes were designated as
p369 and p391, respectively.
The Ig heavy and light chain expression vectors containing 38C13 Id
and a Cla I/Not I cloning cassette at the end of the CH3 exon were
constructed as previously described.10 To construct the Id-CTLA4 fusion
protein, we used overlap polymerase chain reaction (PCR) to replace the
signal sequence of the human CTLA-4 gene with a sequence encoding the
GGGGSGGGGS peptide linker and introduced a stop codon upstream of
the transmembrane domain. The forward primer 5⬘-GGAGGCGGGGGCTCGATGCACGTGGCCCAGCCTG-3⬘ and the reverse primer 5⬘GGATCGCGGCCGCTCAGTCAGAATCTGGGCACGGTTC-3⬘ were used
in the first round PCR on the human CTLA-4 gene. The resulting PCR
product was used as template to perform a second round PCR using the
same reverse primer and a second forward primer 5⬘-CTTATCGATGGAGGCGGGGGCTCGGGAGGCGGGGGCTCG-3⬘. The 5⬘ side of the first
forward primer and the 3⬘ side of the second forward primer have a 15
nucleotide overlap. The final PCR product was ligated into pCR-Blunt
vector (Invitrogen, San Diego, CA) to produce plasmid pBlunt-CTLA4 for
sequencing. To create a mutant CTLA-4 protein, we used the Quick Change
Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA) to introduce a
point mutation at CTLA-4 residue 104 (Tyr3Ala) in pBlunt-CTLA4. A pair
of overlapping primers, 5⬘-CCACCGCCAGCCTACCTGGGC-3⬘ and 5⬘GCCCAGGTAGGCTGGCGGTGG-3⬘, was used to create the point mutation. The pBlunt plasmids were digested at the Cla I and Not I sites, which
were introduced in the PCR primers, to release the DNA fragments
containing the wild-type or mutant CTLA-4 gene. The resulting fragments
were inserted into the heavy-chain plasmid at the end of the CH3 exon. The
resulting plasmids, p362 and p497, contain the coding regions for 38C13 Id
joined to the complete human ␥1 constant region, followed by a GGGGSGGGGS linker and the extracellular domain of CTLA-4 or CTLA-4Y104A,
respectively.
Production and purification of idiotype fusion proteins
The heavy-chain vectors p362 and p497 were cotransfected with the
light-chain vector p307710 into an Ig-nonproducing plasmacytoma cell line,
FO, by electroporation. Transfectomas were selected by resistance to G418
and the highest producer was selected for protein production. Transfectoma
clones were expanded for large-scale production in Dulbecco modified
Eagle medium (DMEM) containing 1% low IgG fetal calf serum (FCS)
(HyClone Laboratories, Logan, UT) and purified by protein A chromatography as previously described.22
Transfection of Chinese hamster ovary cells
Stable transfectants expressing B7-1 or B7-2 were isolated following
electrotransfection of DG44 cells with plasmids p369 and p391, respectively. Electroporation was performed at 210 V, 400 ␮F, 13 ohm.
Transfectants were selected by resistance to G418 (0.5 mg/mL) and
screened for expression of B7 molecules by fluorescence-activated cell
sorting (FACS) analysis. Lines expressing high levels of B7 (CHO/B7-1,
CHO/B7-2) were isolated by sorting on a FACStarPlus (Becton Dickinson,
Mountain View, CA).
Flow cytometry analysis
To screen for expression of B7-1 or B7-2, transfected CHO cells were
removed from their culture vessels by incubation in phosphate-buffered
saline (PBS) containing 10 mmol/L EDTA. Cells were reacted with
biotin-conjugated hamster antimouse B7-1 (16-10A1, PharMingen, San
Diego, CA) monoclonal antibody (mAb) at 1:800 or rat antimouse B7-2
(GL1, PharMingen) mAb at 1:800. The bound biotinylated Abs were
detected with fluorescein isothiocyanate (FITC)-labeled avidin (Cappel,
ICN Biomedicals, Costa Mesa, CA) at 1:500 and analyzed on a FACSCallibur (Becton Dickinson).
To detect the reactivity of various Id proteins to DC2.4 and B7
transfected CHO cells (CHO/B7-1, CHO/B7-2), 5 ⫻ 105 cells were reacted
at 4°C for 30 minutes with 20 ␮g/mL of each Id fusion protein. The bound
Id proteins were detected with FITC-labeled goat antihuman ␬ Ab (Cappel)
at 1:200 and subjected to FACS analysis.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and immunoblot analyses
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
and transfer of proteins to nitrocellulose membrane by semidry electroblotting were performed as previously described.23 Blots were probed with
either a biotinylated anti-Id mAb, S5A8 (IgG2b)24 at 1:1000, or mouse
antihuman CTLA-4 mAb (BNI3; IgG2a; PharMingen) at 1:500. The bound
biotinylated Abs were then reacted with horseradish peroxidase (HRP)conjugated avidin (Cappel) at 1:2000 and detected with the enhanced
chemiluminescence (ECL) system (Amersham, Arlington Heights, IL),
according to the manufacturer’s directions.
Immunization
Mice were immunized 2 times at 2-week intervals by intraperitoneal or
subcutaneous injection of an appropriate amount of Id fusion proteins in
200 ␮L PBS. For some experiments, mice were immunized twice at 2-week
intervals with a mixture of 2 ␮g hepatitis B surface Ag (HBsAg) and 10 ␮g
of the various Id proteins. Immune sera were collected right before and 2
weeks after the booster immunization.
Enzyme-linked immunosorbent assay
Serum samples were collected by tail bleeding. Anti-Id and anti-HBsAg
levels in the immune sera were quantitated by titering sera on enzymelinked immunosorbent assay (ELISA) plates coated with purified 38C13 Id
or yeast-derived recombinant HBsAg, respectively. Bound proteins were
detected with HRP-conjugated goat antimouse IgG Fc (1:1000; Cappel).
Color was generated by adding 2,2⬘-azino-bis(ethylbenzthiazoline sulfonic
acid) (Sigma Chemical, St Louis, MO), and the absorbance at 405 nm was
measured on an ELISA reader. For measurement of IgG anti-Id Abs, the
readings were referenced to a mixture of purified monoclonal anti-38C13 Id
Abs containing IgG1, IgG2a, and IgG2b isotypes24 in a 2:1:1 ratio. For
measurement of IgG anti-HBsAg Abs, the readings were referenced to a
standard serum pooled from 5 mice given injections of 2 ␮g recombinant
HBsAg with complete Freund adjuvant, and the results were expressed as
arbitrary units per milliliter (1 U ⫽ 50% maximum optical density).
Lymphocyte proliferation assay
One week after the booster immunization, splenocytes were collected and
passed through a nylon wool column to enrich T lymphocytes. To perform
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
IDIOTYPE-CTLA4 FUSION PROTEIN AS A CANCER VACCINE
3665
the lymphoproliferative assay, 100 ␮L of 2 ⫻ 106 cells was added to each
well in 96-well flat-bottomed plates and stimulated with 50 ␮g/mL 38C18
Id or transferrin (Sigma). Cells stimulated with 5 ␮g/mL concanavalin A
served as a positive mitogenic control. Negative control wells received cells
only. After 7 days in culture, the cells were pulsed with [3H]thymidine (0.5
␮Ci/well) for 16 hours and incorporated radioactivity was determined using
Top Count (Packard, Meriden, CT). The stimulation index was calculated as
the mean counts per minute of the stimulated wells divided by the mean
counts per minute of the negative control wells.
Tumor challenge
Mice were challenged intraperitoneally with 200 38C13 cells or subcutaneously with 1000 38C13 cells 2 weeks after the booster immunization. It was
previously shown that these are lethal inocula that killed all unprotected
mice. For subcutaneous challenge, tumors were measured every other day,
and the tumor volume (in cubic millimeters) was approximated using the
ellipsoidal formula: length ⫻ width ⫻ height ⫻ 0.52. The mean volume and
SD of mice bearing measurable tumor mass in each group were calculated.
Animals were killed when subcutaneous tumors measured more than 3000
mm3 or until any mouse appeared to be moribund. For intraperitoneal
challenge, the survival of challenged mice was followed and results were
analyzed for significance by the Student t test. Data were considered
statistically significant at P ⱕ .05.
Results
Construction of Id-CTLA4 fusion protein
The genetic constructs to make heavy and light chains of the
chimeric Id protein were previously reported.10 Briefly, the lightchain expression vector contains the 38C13 light-chain variable
region gene joined to the human ␬ constant region gene. The
heavy-chain expression vector contains the 38C13 heavy-chain
variable region gene joined to the human ␥1 constant region gene.
The resulting Id protein retained the original Id determinants
expressed on the native murine 38C13 Id protein. To make the
Id-CTLA4 fusion protein, a PCR fragment encoding a flexible
linker (GGGGSGGGGS) and the extracellular domain of human
CTLA-4 was ligated to the end of the CH3 exon of the heavy-chain
gene. This modified Id heavy-chain vector was cotransfected with
the Id light-chain vector to make Id-CTLA4.
The Id-CTLA4 fusion protein is expected to consist of a
mouse–human chimeric molecule with CTLA-4 attached to each of
the carboxy-terminal ends of the heavy chain, as shown in Figure
1A. SDS-PAGE and immunoblotting techniques were applied to
analyze the purified Id, the fusion protein Id-CTLA4, and a
previously described fusion protein consisting of Id and murine
GM-CSF (Id-GM).10 Under reducing conditions, the light chains of
the Id and both Id fusion proteins all migrated at an apparent
molecular weight of 28 kd (Figure 1B, lanes 4-6). However, the
heavy chains of Id-CTLA4 migrated at an apparent molecular
weight of 74 kd, which is similar to the 75-kd heavy chain obtained
from Id-GM but not the smaller heavy chain (60 kd) from Id
protein, indicating that the Id-CTLA4 fusion protein contained the
CTLA-4 tail. This speculation was confirmed by an immunoblot
analysis that showed that a human CTLA-4–specific Ab recognized
the Id-CTLA4 fusion protein but not Id or Id-GM (Figure 1C, lanes
4-6). The heavy and light chains of the Id and both Id fusion
proteins were properly assembled to give tetrameric proteins, as
observed on a nonreducing gel (Figure 1B, lanes 1-3). However,
unlike the Id protein, which was secreted as a single molecule,
Id-CTLA4 and Id-GM migrated as doublets in the nonreducing gel.
The double bands of the fusion proteins were also present in the
Figure 1. Construction of tumor Id and CTLA-4 fusion protein. (A) Schematic
diagram of Id-CTLA4. Solid areas represent variable regions from the 38C13 tumor.
Open areas represent human ␥1 and ␬ constant regions. Checkered regions
represent the human CTLA-4 sequence. (B) SDS-PAGE of Id (lanes 1 and 4), Id-GM
(lanes 2 and 5), and Id-CTLA4 (lanes 3 and 6) under nonreducing (lanes 1-3) or
reducing (lanes 4-6) conditions. The molecular weight is determined by marker
proteins. (C) Immunoblot analysis of Id fusion proteins. Id (lanes 1 and 4), Id-GM
(lanes 2 and 5), and Id-CTLA4 (lanes 3 and 6) under nonreducing (lanes 1-3) or
reducing (lanes 4-6) conditions were subjected to SDS-PAGE followed by electroblotting to nitrocellulose membrane. The strips were reacted with S5A8, a monoclonal
anti-38C13 Id (lanes 1-3) or mouse antihuman CTLA-4 (lanes 4-6) Abs and detected
with HRP-conjugated second-step reagents.
reducing gel (Figure 1B, lanes 5 and 6), suggesting that they were
the result of proteolytic cleavage of CTLA-4 of Ig and GM-CSF of
Ig, or perhaps due to the involvement of glycosylation.
Reactivity of Id-CTLA4 with B7-1 and B7-2
To investigate the functional activity of Id-CTLA4, we first tested
its binding to a murine dendritic cell line, DC2.4, which expresses
high levels of B7-1 and B7-2.19 As shown in Figure 2A, the
Id-CTLA4 fusion protein clearly demonstrated its ability to bind to
DC2.4 cells. We ruled out the possibility that interaction of
Id-CTLA4 and DC2.4 cells was through the Fc␥ receptor because
under the same condition the Id and Id-GM did not show significant
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3666
HUANG et al
Figure 2. Id-CTLA4 retains the binding activity for B7-1 and B7-2. (A) DC2.4, a
murine dendritic cell line, was stained with Id, Id-GM, or Id-CTLA4. (B) CHO (panels
i,iv, and vii), CHO/B7-1 (panels ii,v, and viii), and CHO/B7-2 (panels iii,vi, and ix) cells
were stained with Id (i-iii), Id-GM (iv-vi), or Id-CTLA4 (vii-ix). Cells were then washed
and incubated with FITC-conjugated goat antihuman ␬ Ab. Dashed lines represent
fluorescence from cells without addition of the tested Id fusion proteins.
binding to DC2.4 cells. We also used B7-1– or B7-2–transfected
CHO cell lines in FACS analysis to directly demonstrate the
binding activity of Id-CTLA4. As shown in Figure 2B, Id-CTLA4
was bound by CHO/B7-1 and CHO/B7-2, but not by untransfected
CHO cells. In contrast, no binding of Id and Id-GM was detected
with either CHO/B7-1 or CHO/B7-2 cells. Thus, the Id-CTLA4
fusion protein retains the binding activity for both of its counter
receptors on APCs.
Immune responses induced by Id-CTLA4 fusion protein
An initial experiment was designed to compare the immunogenicity of Id-CTLA4 with Id-GM, which was previously shown to be an
excellent immunogen.10,11 Mice were immunized subcutaneously
or intraperitoneally with 50 ␮g of Id, Id-CTLA4, or Id-GM and
boosted 2 weeks later with the same amount of Ag. After a single
immunization, the anti-Id Ab titers induced by Id-CTLA4 were
comparable to those obtained by Id-GM (Figure 3A). Immuniza-
Figure 3. Anti-Id titer induced by immunization with Id fusion proteins. Mice
were immunized twice by subcutaneous or intraperitoneal injection of 50 ␮g of Id,
Id-GM, or Id-CTLA4, and bled 2 weeks after each immunization. Anti-Id titers in sera
after the first (A) or second (B) immunization were determined by ELISA, as
described in “Materials and methods.” The data are presented as the mean ⫾ SD for
5 animals of each group.
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
tion with Id alone barely induced any detectable anti-Id Abs in the
primary reaction. Following one booster immunization, a further
increase of anti-Id titers was observed in mice that received
Id-CTLA4 or Id-GM (Figure 3B). High levels of anti-Id Abs were
present in every immunized animal of these 2 groups. The booster
immunization also slightly increased the anti-Id Ab responses in
the Id-immunized group but the titers remained low. Analysis of
sera from each animal in this group revealed that 60% of animals
receiving Id by intraperitoneal immunization remained seronegative, whereas all animals in the subcutaneous group had seroconverted. We measured anti-Id isotypes in the sera of mice treated
with Id, Id-CTLA4, or Id-GM. Regardless of the type of Id proteins
used for vaccination or the route of Ag delivery (subcutaneous or
intraperitoneal), the predominance of IgG1 over IgG2a was observed in all animals (data not shown). The specificity of the anti-Id
antibodies induced by the Id fusion proteins was confirmed by their
reacting with 38C13 tumor cells but not with V1-1 (Id negative) or
V2 (Ig negative) variant cells (data not shown).
To determine the minimal amount of Id-CTLA4 necessary for
induction of Ab responses, the animals were given 2 injections of
various doses (10, 2, 0.5, or 0.1 ␮g) of Id-CTLA4 and the anti-Id
Ab levels were assayed at 2 weeks after injection. Mice immunized
with 10 or 2 ␮g Id served as controls. As shown in Figure 4, mice
immunized with Id alone at a dose of either 10 or 2 ␮g produced
only low titers of anti-Id Ab. In contrast, as little as 0.1 ␮g
Id-CTLA4 was effective in inducing high titers of anti-Id Ab. In
fact, the anti-Id titer induced by 0.1 ␮g Id-CTLA4 was comparable
to titers induced by up to 2 ␮g Id-CTLA4 and was only a little
lower than that induced by 10 ␮g Id-CTLA4 (Figure 4). These
results clearly demonstrate that the vaccine efficacy in terms of
dosage used in immunization can be dramatically increased by
fusing Id to the CTLA-4 molecule.
The enhancing effect of Id-CTLA4 on T cells was then
examined. Mice were intraperitoneally immunized with Id or
Id-CTLA4 and boosted 2 weeks later with the same Ag. Mice that
received PBS served as negative controls. At 1 week after the
second immunization, splenocytes were examined for proliferation
in response to specific Ag stimulation. Splenic lymphocytes
derived from Id-immunized animals did not respond to 38C13 Id
stimulation, with a stimulation index of about 3.0, which was not
Figure 4. Effect of Id-CTLA4 dose on anti-Id titer. Mice were immunized twice
subcutaneously with various doses of Id or Id-CTLA4 and bled 2 weeks after the
second immunization. Anti-Id titers in immune sera were determined by ELISA. The
data are presented as the mean ⫾ SD for 5 animals of each group.
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much different from that produced by splenocytes from PBScontrol animals (Figure 5). In contrast, Id-CTLA4 fusion protein
enhanced the cellular proliferation, with the stimulation index
increasing to about 12.4. All mice failed to respond to transferrin
included as a control Ag. These results demonstrated that immunization with Id-CTLA4 fusion protein also induced the Id-specific
T-cell arm of the immune response.
Tumor protection associated with Id-CTLA4 immunization
The various Id proteins were next compared for their efficacy in
inducing protection against a lethal tumor challenge. Mice were
subcutaneously immunized twice with 50 ␮g Id, Id-CTLA4, or
Id-GM and challenged intraperitoneally with 38C13 tumor cells 2
weeks following the second immunization. The control mice
(injection of PBS alone) or mice immunized with Id did not show
any protection; all of these mice died within 4 weeks of the tumor
challenge (Figure 6A). In contrast, mice immunized with IdCTLA4 significantly suppressed tumor growth and resulted in 80%
long-term survivors (P ⬍ .025 versus medium control group),
which was comparable to the 70% protection rate achieved by
Id-GM (P ⬍ .05 versus medium control group). These long-term
survivors remained disease free for an observation period of 120
days. In a separate experiment, mice were intraperitoneally immunized with 10 ␮g Id and Id fusion proteins and subcutaneously
inoculated with 38C13 tumor cells. Compared with the PBS control
group, Id-CTLA4 and Id-GM immunization resulted in 40% (2 of 5
mice) and 60% (3 of 5 mice) tumor-free animals (⬎ 60 days),
whereas all animals in the control group had detectable tumors by
day 17. In addition, objective tumor growth suppression was
observed in tumor-bearing animals in the Id-CTLA4 and Id-GM
groups (Figure 6B). By day 24, the mean tumor volume of the
tumor-bearing animals in the Id-CTLA4 and Id-GM groups was
265 ⫾ 99 mm3 and 221 ⫾ 142 mm3, respectively, as compared
with 2140 ⫾ 771 mm3 in the PBS-control group. Immunization
with Id alone led to tumor suppression to some extent; however, no
tumor-free animals were observed in this group.
Figure 6. Survival of mice challenged with 38C13 tumor cells. (A) C3H/HeN mice
were immunized twice subcutaneously with 50 ␮g of the tested Id fusion proteins or
PBS alone and intraperitoneally challenged with 38C13 cells 2 weeks after the
second immunization. The percentage of survivors in each group was recorded.
(B) C3H/HeN mice were immunized twice intraperitoneally with 10 ␮g of the tested Id
fusion proteins or PBS alone and subcutaneously challenged with 38C13 cells 2
weeks after the second immunization. The mean tumor volume of mice bearing a
measurable tumor mass in each group was calculated. SD (bars) are only given at
day 24 for clarity. The ratio of animals in each group that succumbed to tumor death
is indicated.
Mechanisms involved in the high immunogenicity of Id-CTLA4
Figure 5. Id-specific T-cell responses induced by Id-CTLA4 immunization.
C3H/HeN mice were immunized twice intraperitoneally with 10 ␮g Id or Id-CTLA4.
Mice receiving PBS only served as controls. Splenocytes pooled from 3 immunized
mice were stimulated with 50 ␮g 38C13 Id (䡺) or transferrin (f). Values are
presented as mean stimulation index for triplicate wells ⫾ SD. The mean counts per
minute for the mitogenic control (5 ␮g/mL concanavaline A) of the PBS, Id, and
Id-CTLA4 groups were 32 305 ⫾ 4196, 25 088 ⫾ 3123, and 51 125 ⫾ 4307, respectively. The mean counts per minute for the negative control wells (receiving cells only)
of the PBS, Id, and Id-CTLA4 groups were 2151 ⫾ 372, 1105 ⫾ 168, and 1922 ⫾ 274,
respectively.
We next asked whether the biologic activity of CTLA-4, that is, the
ability to bind B7 molecules on APCs, was required for the
enhanced immunogenicity of Id-CTLA4. We constructed an Id
fusion protein containing a mutant form of human CTLA-4
(CTLA4Y104A) with a point mutation at residue 104 (Tyr3Ala) in
the MYPPPY motif (residue 99-104). The MYPPPY motif located
in the CDR3-like region of CTLA-4 is critical for its binding to B7
molecules, and the mutation of Tyr 104 in this region to Ala
completely abolishes the interaction of the mutant CTLA-4 to both
B7-1 and B7-2.25 The purified Id-CTLA4Y104A retained the idiotypic determinant as demonstrated by its interaction with the
specific anti-Id mAb (data not shown). In FACS analysis using
B7-1– or B7-2–transfected CHO cell lines, we confirmed that
Id-CTLA4Y104A lost its binding activity to both B7-1 and B7-2
molecules (data not shown). We then intraperitoneally immunized
mice with 10 ␮g Id, Id-CTLA4, or Id-CTLA4Y104A and analyzed
the presence of specific anti-Id Ab in the immune sera. As shown in
Figure 7, mice receiving 2 Id-CTLA4Y104A inoculations produced
only low titers of anti-Id Ab similar to the titers achieved by Id
immunization, whereas a single immunization with Id-CTLA4 was
able to produce high titers of anti-Id Ab. When the mice were
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3668
HUANG et al
injected intraperitoneally with a lethal dose of 38C13 tumors, none
of the animals in the Id and Id-CTLA4Y104A groups survived the
challenge, whereas immunization with Id-CTLA4 resulted in 60%
long-term survivors. Taken together, these results strongly suggest
that the interaction of CTLA4 and B7 on APCs is required for the
enhanced antitumor immunity of Id-CTLA4.
We further investigated whether the physical linkage between
Ag and CTLA-4 was important for the adjuvant effect of CTLA-4.
We mixed Id, Id-GM, or Id-CTLA4 with a model Ag, in this case
HBsAg, and twice intraperitoneally injected the mixture into mice.
Although much stronger anti-Id Abs were generated in the HBsAg
plus Id-GM and HBsAg plus Id-CTLA4 groups compared with
those generated in the HBsAg plus Id group (Figure 8A), all groups
produced similar levels of anti-HBsAg Abs (Figure 8B). Thus, the
CTLA-4 or cytokine molecules must be physically linked to the Ag
to achieve increased immune responses.
Discussion
Most tumor malignancies express tumor-associated or tumorspecific Ags but do not elicit an efficient immune response. Various
experimental strategies have been explored to enhance the immunogenicity of tumor vaccines. In the present study, we used B-cell
lymphoma as a model system to test a new strategy for inducing
antitumor immunity by specifically targeting APCs with fusion
proteins consisting of tumor-derived Id protein and CTLA-4. We
found that the Id-CTLA4 fusion protein elicited strong Id-specific
T-cell responses and anti-Id Abs that specifically bind to Id-positive
tumor cells. Mice immunized with Id-CTLA4 were significantly
protected against a lethal tumor challenge. The immunogenicity of
Id-CTLA4 is so potent that as little as 0.1 ␮g induced high titers of
anti-Id Abs.
Previous experiments have shown that targeting immunogens to
various APC populations with Abs against class II MHC,13,14
Fc␥R,13 33D1,14 or surface Ig15 can substantially increase Ab
responses. Immunoconjugated Ag was demonstrated to be more
efficiently presented to T cells than Ag alone in an in vitro assay.26
Compared to the previous studies using immunogenic proteins as
Figure 7. Reduction of anti-Id Ab response by immunization with Id-CTLA4Y104A
mutant protein. Mice were immunized twice by intraperitoneal injection of 10 ␮g Id,
Id-CTLA4Y104A, or Id-CTLA4 and bled 2 weeks after each immunization. First
immunization, f; 2nd immunization, 䡺. Anti-Id titers in immune sera were determined
by ELISA, as described in “Materials and methods.” The data are presented as the
mean ⫾ SD for 5 animals in each group.
BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
Figure 8. Coimmunization of mice with HBsAg and various Id proteins. Mice
were immunized twice intraperitoneally with a mixture of 2 ␮g HBsAg and 10 ␮g Id,
Id-GM, or Id-CTLA4. Sera were collected 2 weeks after the second immunization.
Anti-Id (A) and anti-HBsAg (B) titers were determined by ELISA, as described in
“Materials and methods.” The data are presented as the mean ⫾ SD for 5 animals in
each group.
Ags, we found that fusion with CTLA-4 was able to convert a lowto nonimmunogenic tumor Ag into a strong immunogen. CTLA-4
likely provides such a strong adjuvant activity due to its strong
binding affinity to B7 molecules on dendritic cells. Among the
professional APCs, dendritic cells express high levels of both B7-1
and B7-2 and are particularly important in initiating primary
immune responses.27 CTLA-4 binds to both B7-1 and B7-2 with a
20- to 50-fold higher affinity than CD28,28 another B7-binding
receptor on T cells. Using flow cytometric analysis, we found that
the Id-CTLA4 fusion protein retained its ability to bind to both
B7-1 and B7-2 molecules (Figure 2B) and interacted strongly with
a murine dendritic cell line (Figure 2A). Another feature of
Id-CTLA4 that might contribute to its strong binding to B7expressing APCs is the presence of 2 CTLA-4 molecules in its
construct. The dimeric CTLA-4 in the Id-CTLA4 fusion protein
can bind 2 B7 molecules29 and thus increase its binding avidity.
Another possible explanation for the high immunogenicity of
Id-CTLA4 involves the carrier effect of the human Ig constant
region or human CTLA-4 used to construct the fusion protein.
Indeed, we observed anti-Id Ab responses in mice that received the
Id protein, although the magnitude was much weaker than responses induced by Id-CTLA4 or Id-GM (Figures 3 and 4). We also
noted the presence of antihuman IgG Ab in all immunized groups
and antihuman CTLA-4 Ab in the group immunized with IdCTLA4 (data not shown). In a previous study, it was also reported
that the xenogenic human IgG constant region was required for a
DNA vaccine to induce anti-Id responses.30 However, for several
reasons we believe that the contribution of xenogeneic carrier
effect to the immunogenicity of Id-CTLA4 observed in our study
was minimal. First, the presence of a xenogenic determinant is not
absolutely required for creating immunogenicity of the Id protein.
One example is that recombinant Id proteins containing only the
lymphoma Ig variable region and chemokines were able to elicit
high levels of specific anti-Id Abs and protective immunity.12
Second, we previously made an Id fusion protein containing the
xenogenic human Ig constant region and human GM-CSF that has
a size similar to Id-CTLA4, but that has no GM-CSF activity in
mice.10 In contrast to the high immunogenicity of Id-CTLA4, the
Id-human GM-CSF fusion protein failed to produce anti-Id Abs.
This finding strongly suggests that the contribution of the xenogenic carrier protein to the immune-enhancing function of IdCTLA4 is minimal; instead, the biologic activity of CTLA-4, that
is, the ability to bind B7 molecules, is critical for its enhanced
immunogenicity. To more clearly address this issue, we constructed
a mutant Id-CTLA4 fusion protein (Id-CTLA4Y104A) that has no
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BLOOD, 1 DECEMBER 2000 䡠 VOLUME 96, NUMBER 12
IDIOTYPE-CTLA4 FUSION PROTEIN AS A CANCER VACCINE
binding activity to either B7-1 or B7-2 molecules. We demonstrated that Id-CTLA4Y104A, like the control Id protein, produced
much less anti-Id Ab than Id-CTLA4 (Figure 7) and failed to
provide protection against 38C13 tumor challenge. Together, these
results indicate that the strong adjuvant activity of Id-CTLA4 is
largely due to its more efficient targeting to APCs but not the
classic carrier effect of the xenogenic Ig or CTLA-4 in the
fusion protein.
In several previous studies using Id-cytokine or Id-chemokine
fusion proteins as vaccines, it was demonstrated that vaccine
efficacy depended on covalent linkage of the Id protein and
cytokine or chemokine.10-12 In the present study, we performed an
experiment coimmunizing a stronger Ag (HBsAg) with Id-CTLA4
and saw no enhancement of immune responses to HBsAg (Figure
8B). Therefore, the physical linkage between Id and CTLA-4 is
likely required for the increased immunogenicity of Id-CTLA4. We
hypothesize that the presence of CTLA-4 moiety in the fusion
protein would ensure its targeting to APCs and, therefore, leads to
more efficient uptake and processing of Id-CTLA4. This assumption is supported by a recent study that a CTLA4-Ig fusion protein
composed of CTLA-4 and IgG Fc was present at 4-fold higher
levels in draining lymph nodes than a nontargeted protein within 24
hours of administration.31
In contrast to the enhanced immunogenicity of Id-CTLA4
found in the present study, CTLA4-Ig was reported to suppress
formation of Ab responses to sheep red blood cells and keyhole
limpet hemocyanin.32 CTLA4-Ig has also been used as an immunosuppressive drug in animal models of transplantation and autoimmune diseases.33,34 The immune suppression mediated by CTLA4-Ig
is likely through its inhibition of CD28-B7 interactions, which
provide an important positive signal for T-cell proliferation and
cytokine release.35 The discrepancy between the immunomodulating effects of CTLA-4 in our study and in previous studies is likely
due to the number of treatment doses of CTLA-4 administered. In
the previous studies, to achieve maximal immune suppression, a
high dose (50-500 ␮g) of CTLA4-Ig was applied before Ag
treatment and was continued several days thereafter. In our study,
Id-CTLA4 was administered only twice with a 2-week interval
between immunizations and at significantly low doses. Using our
protocol, it is likely that the B7 molecules on APCs are not
completely blocked, even at a relatively high dose of 50 ␮g, and
thus remain available to engage with CD28 and thereby provide a
signal for T-cell activation. Indeed, we found that mice immunized
with Id-CTLA4 elicited Id-specific and human Ig-specific T-cell
proliferation responses (Figure 5 and data not shown). Vaccination
3669
with DNA encoding CTLA4-Ig provides additional evidence that
CTLA4-Ig at low levels increases both Ab and T-cell proliferation
responses.16,36 Another possible strategy to overcome the potential
immune-suppressive effect of CTLA-4 may be to apply a mutant
CTLA-4 (CTLA4Y100A). The mutation of the first Tyr to Ala at
residue 100 in the important MYPPPY motif completely abolishes
CTLA-4 binding to B7-2, whereas its B7-1 reactivity is retained.28
Thus, the Id-CTLA4Y100A fusion protein would be directed to APCs
through interaction with B7-1, whereas the remaining B7-2 molecules on APCs would serve to activate T cells.
Other immune-enhancing molecules used to increase the immunogenicity of Id in the fusion context include cytokines (GM-CSF,
IL-1␤, IL-2, IL-4),10,11,30,37 chemokines (IP-10, monocyte chemotactic protein 3),12 as well as a nontoxic fragment of tetanus toxin.38
Among these, the adjuvant effect of GM-CSF is unique and the best
characterized. GM-CSF is known to be a critical stimulatory
cytokine for a variety of APCs by increasing expression of class II
MHC, B7-1, and B7-2, as well as other adhesion molecules.39-41
Recombinant GM-CSF was shown to provide strong adjuvant
activity to Id proteins in animal studies42 and human clinical
trials.43 We demonstrated in this study that Id-CTLA4 and Id-GM
were almost identical in their ability to induce primary and
secondary anti-Id Ab responses, as well as protective immunity.
Because the adjuvant effects of Id-CTLA4 and Id-GM appeared to
work on APCs but are dependent on different mechanisms, the
combination of these 2 fusion proteins in vaccine formulation
might have additive effects. Studies in this regard are currently
under investigation in our laboratory.
In summary, we demonstrated that fusion of CTLA-4 to a weak
Id Ag dramatically increases its immunogenicity and substantially
promotes specific Ab responses and protective immunity. This
approach is simple and does not require the use of immunologic
adjuvants. The general approach of CTLA-4 fusion vaccines
may be applicable to tumor Ags of other cancers as well as
infectious diseases.
Acknowledgments
We thank Drs Sherie L. Morrison (UCLA, Los Angeles, CA) and
Chou-Chik Ting (National Cancer Institute, National Institutes of
Health, Bethesda, MD) for critical reading of this manuscript and
many helpful suggestions. We also thank Mr Douglas Platt for the
English editing of this manuscript.
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2000 96: 3663-3670
Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor
antigen
Tzu-Hsuan Huang, Pin-Yi Wu, Chin-Nien Lee, Hsing-I Huang, Shie-Liang Hsieh, John Kung and
Mi-Hua Tao
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