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Unité de Biologie des Régulations Immunitaires and
Unité de Chimie Organique, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Tumor-associated carbohydrate Ags are also potential targets for anti-cancer therapy (10). Among these Ags, T, Tn, sialyl-Tn, and the ganglioside GM2 are the most well known. Covalent attachment of the carbohydrate Ags to protein carriers is traditionally used to induce anti-cancer Ab responses, and the resulting immunogens were shown to increase survival in patients. The most promising beneficial effects of tumor vaccination using this strategy were found for the GM2/keyhole limpet hemocyanin (KLH)3 conjugate against melanoma (11) or the sialyl-Tn/KLH conjugate against breast, ovarian, and colorectal cancers (12).
Recently, we developed a fully synthetic immunogen that does not require protein carrier (13, 14). This system, called multiple antigenic glycopeptide (MAG), is based on a dendrimeric lysine core with four arms analogous to the multiple antigenic peptide (MAP) construct of Tam (15). Each arm is linked to a peptide backbone containing a CD4+ T cell epitope (PV peptide) with a monomeric saccharide Tn residue at the N-terminal end of the peptide (MAG:Tn-PV). This construction offers several advantages: the carbohydrate content is much higher than in traditional protein conjugates, the core matrix is nonimmunogenic, and the construction has a well-defined chemical structure. A therapeutic immunization performed with this immunogen was shown to increase the survival of tumor-bearing mice (14).
Polysaccharidic cancer-associated Ags (GM2, GD2, Globo H,
Ley, Sialyl-Tn) can display large glycotopic
structures available for Ab binding sites. In contrast, Tn, which is a
monosaccharidic Ag (
-GalNAc-Ser/Thr) found on mucin-type
glycoproteins and expressed on most human adenocarcinomas
(16), was shown to be recognized by different mAbs as Tn
clusters (17, 18, 19, 20). Recent encouraging results obtained
with a linear glycopeptide based on a tri-Tn glycotope recognized by
the MLS128 mAb showed that anti-Tn Abs can be successfully induced
in mice in the absence of a carrier protein (21). To
further improve our MAG vaccines, the introduction of a cluster of
three Tn was undertaken to mimic native sources of Tn on tumor
cells.
We present here the results of both prophylactic and therapeutic vaccinations using a MAG construct with a lysine core carrying four copies of the PV peptide further extended with a tri-Tn glycotope (MAG:Tn3-PV). In both cases, the MAG:Tn3-PV afforded good protection against the development of Tn-expressing tumor cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The Tn Ags (-GalNAc-Ser/Thr) were synthesized by classical
methods (22, 23). Synthesis of the MAG:Tn-PV, MAG:Tn3-PV,
MAP:PV, MAP:PV2, and Tn6-PV was performed by solid phase methodology
using Fmoc chemistry as described previously (13, 21). After attachment of the 
-alanyl spacer on the Wang
resin the lysine core was assembled by coupling successively two levels
of FmocLys-(Fmoc)-OH, providing four amino groups. The lysine core was
further elongated by the amino acids of the T epitope sequence of the
poliovirus (KLFAVWKITYKDT) (24) to produce the MAP:PV. The
glycosylated amino acid -GalNAc-Thr-OH was then introduced on the
four branches as its pentafluorophenyl ester in the presence of
N-hydroxybenzotriazole (21). The incorporation
was repeated, and ultimately -GalNAc-Ser-OH (pfp ester) was coupled
to the construction. After deprotection and cleavage from the resin the
products were purified by HPLC using a Perkin-Elmer pump system (Foster
City, CA) with a UV detector at 230 nm; the column was a Nucleosil
C18 (5 µm, 300Å, 250 x 10 mm), and the
gradient was performed with water (0.1% trifluoroacetic
acid)/acetonitrile over 20 min (MAG:Tn3-PV: gradient from 5 to 60%;
retention time, 12 min; amino acid analysis: Ala 3.75 (4),
Asp 4.56 (4), Ile 4.13 (4), Leu 4.04
(4), Lys 16.6 (15), Phe 4.0 (4),
Ser 3.82 (4), Thr 16.07 (16), Tyr 4.6
(4), Val 4.01 (4)).
Mice and immunization
Five- to 8-wk-old female BALB/c mice were obtained from Iffa Credo (St. Germain sur l’Abresle, France). To test immunogenicity of the MAG construct, mice were injected i.p. three times with Ag mixed with alum (Serva, Heidelberg, Germany) at 3-wk intervals. For vaccination experiments, mice received three i.p. injections of Ag mixed with alum or alum alone at 10-day intervals, and 10 days after the last boost, mice were challenged i.p. with 1,000 or 20,000 TA3/Ha cells prepared as described below. Alternatively, mice were challenged with the CT26 cell line (provided by Dr. R. A. Reisfeld, The Scripps Institute, La Jolla, CA) that does not express Tn. In some experiments, mice were treated with GK1.5 (anti-CD4) or H35.17.2 (anti-CD8) mAb (200 µg of mAb on days -1, 0, and 1 at the time of immunization). In all cases sera were collected after each immunization and tested for the presence of anti-Tn Abs by ELISA or FACS.
ELISA
To test the binding of MLS128 (a mouse IgG3 specific for the Tn Ag (25); a gift from Dr. H. Nakada, Kyoto Sangyo University, Kyoto, Japan) to the MAG constructs, an ELISA was performed as previously described (13) by coating Ags at 1 µg/ml in 50 mM carbonate buffer, pH 9.6. Mouse sera were tested for anti-Tn as previously described (21), using the synthetic glycopeptide Tn3-G6K(Biot)G or the nonglycosylated analogue STTG6K(Biot)G as a control. Briefly, the biotinylated peptides at 1 µg/ml were incubated for 1 h at 37°C on streptavidin-coated microtiter plates. Then, serial dilutions of sera were performed, and bound Abs were revealed using goat anti-mouse IgG or IgM peroxidase conjugate (Sigma) and o-phenyldiamine/H2O2 substrates as previously described (13). Plates were read photometrically at 492 nm in an ELISA autoreader (Dynatech, Marnes la Coquette, France). The negative control consisted of naive mouse sera diluted 100-fold. ELISA Ab titers were determined by linear regression analysis, plotting dilution vs absorbance at 492 nm. The titers were calculated to be the log10 highest dilution that gave twice the absorbance of normal mouse sera diluted 1/100. Titers were given as the arithmetic mean ± SD of the log10 titers.
Flow cytometry
Mouse sera were tested at serial dilutions by flow cytometry on two tumor cell lines expressing the Tn Ag, the human Jurkat cell (26), and the murine TA3/Ha cell (27). Cells were first incubated for 30 min with serial dilutions of sera at 4°C in PBS containing 5% FCS and 0.05% sodium azide. Then, cells were incubated 30 min with anti-mouse IgG conjugated to FITC and with an anti-mouse IgM conjugated to PE (Caltag, Burlingame, CA). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and analysis was performed with CellQuest software (Becton Dickinson). The titers were calculated to be the log10 highest dilution of sera that gave twice the geometric mean of fluorescence obtained with cells incubated with secondary reagents alone. For competition assay, 5 x 105 Jurkat cells were incubated with 1 µg/ml of the MLS128 mAb mixed with serial dilution of MAG, control MAP, or asialo-ovine submaxillary mucin (aOSM; given by Dr. E. Osinaga, Facultad de Medicina, Montevideo, Uruguay) for 30 min at 4°C, then the binding of MLS128 to Jurkat cells was revealed with an anti-IgG-FITC. Results are expressed as the percent inhibition of the signal obtained with MLS128 alone.
Anti-tumor immunotherapy
For tumor implantation, the preparation of the murine mammary adenocarcinoma cell line, TA3/Ha, differs from our previously published work in which freshly isolated TA3/Ha from in vivo passages was directly used (14). Here, the TA3/Ha cells were grown by passage on BALB/c mice and were frozen to obtain a homogeneous batch. Two or 3 days before tumor graft, cells were thawed and cultured in vitro in DMEM containing 5% FCS. Cells were then recovered and centrifuged at 500 rpm for 5 min. In these conditions, cells recovered from the pellet were homogeneous in size and included <50% dead cells. Cells were checked for Tn expression by FACS using the MLS128 mAb before implantation. One thousand TA3/Ha live cells were administered i.p. to 5-wk-old BALB/c mice, then mice were s.c. injected several times, alternatively at the tail base and in the neck, with 100 µg of MAG constructs with 1 mg of alum. Survival of treated and untreated mice was followed for >100 days. Statistical analysis of survival curves was performed with StatView software (Abacus Concepts, Berkeley, CA) using the log-rank test.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We previously showed that a dendrimeric MAG containing a
monosaccharidic Tn motif at the N terminus of its four peptidic arms
(MAG:Tn-PV) was able to induce Tn-specific Abs in a T cell-dependent
manner (14). As the Tn Ag is expressed as clusters in
mucin proteins, it should be advantageous to mimic naturally occurring
Tn structures displayed by cancer cells. Moreover, dimeric and trimeric
carbohydrate epitopes associated with protein carrier have already been
shown to be more effective in generating Ab response than the monomeric
analogue (28, 29). Similarly, we showed, using short
synthetic glycopeptides, that a Tn cluster was required to induce an
efficient immune response (21). To improve our MAG, we
have therefore introduced a Tn cluster corresponding to the glycotope
recognized by the MLS128 mAb. The MLS128 is a Tn-specific mAb obtained
after immunizing mice with the human carcinoma cell line LS180
(25), and this mAb was shown to bind to a tri-Tn cluster
((-GalNac)-Ser-(-GalNac)-Thr-(-GalNac)-Thr) on mucin-like
proteins (17, 18). This mAb can also recognize a dimeric
Tn within the (-GalNac)-Ser-(-GalNac)-Thr-Thr sequence, but with
a much lower affinity (30). Although we previously showed
that a linear glycopeptide based on three consecutive
(-GalNac)-D-Ser was able to induce anti-Tn Abs
(21), the lack of contribution of the aglyconic part of
the structure (Ser or Thr residues) to Ab binding is not clearly
established. Therefore, based on the initial
(-GalNac)-Ser-(-GalNac)-Thr-(-GalNac)-Thr sequence described
for MLS128 binding, we synthesized a dendrimeric MAG with four arms
containing the PV CD4+ T cell epitope
(MAG:Tn3-PV; see Table I
and Fig. 1). Two dendrimeric MAP controls were
also synthesized comprising the PV sequence alone (MAP:PV) or the same
construct with an additional STT peptide (MAP:PV2; Table I).
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A). No
MLS128 binding was observed using the control dendrimeric MAP compounds
devoid of Tn (MAP:PV and MAP:PV2). To precisely compare the two MAGs,
we tested by FACS the ability of the two constructs to inhibit the
MLS128 binding to native Tn structures at the surface of the human
Jurkat cell line. In these conditions, MAG:Tn3-PV strongly inhibited
MLS128 binding to Jurkat cells, whereas MAG:Tn-PV did so only weakly
(Fig. 2B). The MAG:Tn3-PV inhibition was as efficient as
that obtained with aOSM.
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We next evaluated the immunogenicity of the MAG:Tn3-PV compound by
immunizing mice with MAG:Tn3-PV, MAG:Tn-PV or control MAP:PV2. Mice
were immunized three times (days 0, 21, and 42) and were bled 1 wk
after each immunization, and then regularly over a 6-mo period. Sera
were tested for IgG and IgM Tn-specific Abs by ELISA using a
biotinylated glycopeptide containing a tri-Tn cluster (Tn3-G6K(Biot)G).
Fig. 3 shows that Tn-specific Abs (both
IgG and IgM) were induced by MAG:Tn3-PV and MAG:Tn-PV, but not by the
control MAP:PV2. However, the Tn-specific response induced by the
MAG:Tn-PV slowly increased after immunization, whereas that induced by
the MAG:Tn3-PV rapidly increased after two injections and then remained
stable for 5 mo. The isotypic analysis of MAG:Tn3-PV-induced Abs shows
that IgG1 Abs were predominant, but sera also contained a large amount
of IgG3 Abs, which traditionally characterize anti-carbohydrate Ab
responses in mice (data not shown). The increase in Tn density in the
MAG did not modify the Th dependency of the anti-Tn Ab response,
because this response was abolished when mice were depleted of CD4 T
cells in vivo, but remained unchanged when CD8 T cells were depleted
(data not shown). These results show that the introduction of a tri-Tn
glycotope, instead of a monomeric Tn, into the MAG strongly increases
the immunogenicity of the Tn Ag and makes the response more
persistent.
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, titration of sera from
MAG:Tn3-PV-immunized mice on murine adenocarcinoma TA3/Ha cells
demonstrates positive binding of both IgM and IgG Abs, as opposed to
the lack of binding of sera from mice immunized with MAP:PV2. Likewise,
the human Jurkat cell was well recognized by Abs induced after
immunization with MAG:Tn3-PV, but not with MAP:PV2. These results show
that native Tn structures on tumor cells are recognized by Abs induced
by a MAG based on a tri-Tn glycotope, indicating that these Abs can
efficiently target Tn-expressing tumor cells.
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We next verified the capacity of MAG:Tn3-PV-induced Abs to
target and reject the highly tumorigenic TA3/Ha adenocarcinoma in vivo.
Mice were left untreated or were vaccinated three times with 10 µg of
MAP:PV or MAG:Tn3-PV in alum or with alum alone. Then, 10 days after
the last boost, mice were challenged with 1,000 TA3/Ha cells, and
survival of mice to the TA3/Ha graft was followed for 100 days. As
shown in Fig. 5A, all
untreated mice died within 30 days following tumor challenge. In
contrast, 80% of mice vaccinated three times with 10 µg of
MAG:Tn3-PV mixed with alum survived to the tumor challenge, whereas
10% of mice survived in control groups receiving MAP:PV mixed with
alum or alum alone (Fig. 5A). The protection afforded by
MAG:Tn3-PV against the TA3/Ha challenge ranged from 70–90% depending
on the dose of MAG used for vaccination (Fig. 5B). In
contrast, MAG:Tn3-PV vaccination did not protect mice against the
Tn-negative carcinoma CT26 cell line, showing the Tn specificity of the
protection afforded by MAG. Prior to the tumor challenge, Tn-specific
IgG were only detected in MAG:Tn3-PV-vaccinated mice (Ab titers ranging
from 5,000 to 100,000 as detected by ELISA; data not shown). When mice
were depleted of CD4 T cells in vivo, no anti-Tn Ab was induced,
and the protection against TA3/Ha tumor challenge was totally
abrogated, whereas CD8 T cell-depleted mice were still protected (Fig. 5C). Together, these results clearly show that the
anti-Tn immune response induced by the MAG:Tn3-PV is able to confer
a high protection rate against a tumor cell line expressing Tn, and
that this protection depends on the induction of anti-Tn Abs
requiring T cell help.
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Preventive vaccination has limited interest when targeting the
immune response to cancer-associated Ags; therefore, cancer vaccination
should prove efficacious toward pre-existing tumor cells and be used as
a therapeutic tool. Using the TA3/Ha tumor model, Fung et al.
(31) showed that ASI treatments based on a
-Gal(1, 2, 3)-GalNAc glycotope conjugated to the KLH (TF-KLH) can
afford a substantial survival rate (25%) of TA3/Ha-bearing mice. Given
the success obtained with the MAG:Tn3-PV vaccination on the survival of
mice challenged with TA3/Ha cells, we next tested its efficacy in
immunotherapeutic protocols. Mice were given 1000 TA3/Ha cells and were
then treated with 100 µg of MAG:Tn3-PV in adjuvant (aluminum
hydroxide or Freund’s adjuvant) or with adjuvant alone. In these
conditions, about 35–40% survival was observed in MAG:Tn3-PV-treated
groups, whereas all mice died in groups treated with adjuvant or left
untreated (Fig. 6A).
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B, CY followed by
immunization with alum alone did not modify the mortality of TA3/Ha
tumor-bearing mice. When mice were given a 10 mg/kg dose of CY,
followed by the MAG:Tn3-PV treatment given with alum on days 2, 5, 11,
and 17, only 37.5% of mice survived. However, when MAG:Tn3-PV
treatment was given after the administration of a 50 mg/kg dose of CY,
the survival of TA3/Ha-bearing mice reached 80% (Fig. 6B).
These results indicate that CY can improve the efficacy of the
MAG:Tn3-PV treatment.
We further compared the efficacy of the MAG:Tn3-PV treatment with
other immunogenic compounds. In a previous work, we showed that a MAG
compound based on a monomeric Tn (MAG:Tn-PV) was able to improve the
survival of TA3/Ha-bearing mice (14). Fig. 6C
shows that ASI treatment performed with the MAG:Tn-PV together with CY
leads to a 30% protection rate of tumor-bearing mice compared with the
60% protection afforded by the MAG:Tn3-PV-based ASI. This result is in
agreement with the compared immunogenicity of both compounds (Fig. 3).
More recently, we showed that a linear glycopeptide containing a T cell
epitope associated with a double tri-Tn glycotope (Tn6-PV) was able to
induce anti-Tn Abs that recognize Tn on tumor cell lines
(21). The advantage of such a linear glycopeptide over the
MAG strategy is that synthesis, yield, and purification are easier to
achieve. When Tn6-PV was used in ASI, only 30% of mice survived to the
TA3/Ha implantation compared with the 75% survival observed in the
MAG:Tn3-PV-treated group (Fig. 6D). In summary, we show that
the efficacy of ASI afforded by MAG is improved when the Tn Ag is
incorporated as a tri-Tn cluster rather than as a mono-Tn moiety, and
that the tri-Tn glycotope is much more efficient when displayed on the
MAG structure compared with a linear glycopeptide.
It is difficult to ensure that the improved immunogenicity and therapeutic effects of the MAG:Tn3-PV compared with the MAG:Tn-PV result from the use of the tri-Tn glycotope and do not result from a clustering effect due to the higher amount of Tn incorporated into the immunogen. However, the results obtained with the linear glycopeptide (Tn6-PV) indicate that the beneficial effects of Tn clustering may be limited if the number of Tn Ag per branch is considered. Moreover, the injection of the same microgram doses of Tn6-PV and MAG:Tn3-PV corresponds to a 1.5-fold greater amount of Tn or tri-Tn in the case of the Tn6-PV, whereas it shows lower efficacy. Therefore, our results suggest that other parameters should be taken into account, for instance the flexibility of the saccharide moiety. Indeed, there is probably much more rigidity in the double tri-Tn cluster structure of the Tn6-PV, whereas the MAG can display a large variety of Tn glycotopes through its four flexible peptidic arms that can better mimic the diversity of natural Tn clusters. These issues are currently under investigation.
Conclusion
Together, our results show that a synthetic multiple antigenic glycopeptide including a saccharide tumor-associated Ag together with an appropriate CD4+ T cell epitope is highly immunogenic and can efficiently allow rejection of implanted tumor cells when used in therapeutic treatment. Our results also provide evidence that the introduction of well-defined glycotope clusters in carbohydrate-based immunogens is important for the induction of an efficient and long-lasting antitumor response.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard Lo-Man, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
3 Abbreviations used in this paper: KLH, keyhole limpet hemocyanin; MAG, multiple antigenic glycopeptide; ASI, active specific immunotherapy; CY, cyclophosphamide; MAP, multiple antigenic peptide; aOSM, asialo-ovine submaxillary mucin.
Received for publication June 22, 2000. Accepted for publication November 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconjugates J. 15:51.
-D-galactopyranosyl)-L-serine and L-threonine. Carbohydr. Res. 79:C1.
-N-acetylgalactosamine-O-serine/threonine (Tn) antigen-mediated cellular immune response for active immunotherapy in mice. Cancer Res. 51:1406.This article has been cited by other articles:
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T. Buskas, S. Ingale, and G.-J. Boons Glycopeptides as versatile tools for glycobiology Glycobiology, August 1, 2006; 16(8): 113R - 136R. [Abstract] [Full Text] [PDF]
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