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[Cancer Research 59, 1520-1524, April 1, 1999]
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[Cancer Research 59, 1520-1524, April 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

A Fully Synthetic Immunogen Carrying a Carcinoma-associated Carbohydrate for Active Specific Immunotherapy1

Richard Lo-Man2, Sylvie Bay, Sophie Vichier-Guerre, Edith Dériaud, Danièle Cantacuzène and Claude Leclerc

Unité de Biologie des Régulations Immunitaires [R. L-M., E. D., C. L.] and Unité de Chimie Organique [S. B., S. V-G., D. C.], Institut Pasteur, 75724 Paris, Cedex 15, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aberrant glycosylation of mucins leads to the exposure of cryptic carbohydrate antigens at the surface of carcinoma cells, which, therefore, represent potent targets for anticancer therapeutic vaccines. To date, the development of immunogens to stimulate immune response to such saccharidic antigens is based on carbohydrate conjugation to carrier proteins. However, these traditional protein conjugates are poorly defined in chemical composition and structure. As an alternative, we synthesized a multiple antigenic O-linked glycopeptide (MAG) carrying the carbohydrate Tn antigen associated with a CD4+ T-cell epitope (MAG:Tn-PV). This fully synthetic immunogen is highly defined in composition and carries a high saccharidic epitope ratio over the entire molecule. The MAG:Tn-PV was able to induce anti-Tn IgG antibodies that recognize human tumor cell lines. A therapeutic immunization protocol performed with this fully synthetic immunogen increased the survival of tumor-bearing mice. Thus, the accurately defined and versatile MAG system represents an efficient strategy to induce carbohydrate-specific antitumor immune responses but may also be applicable to the prevention of infectious diseases, if it is based on bacterial oligosaccharides.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T and Tn carbohydrate epitopes are found not only on a variety of epithelial cells derived from breast, pancreatic, and colon cancers but also on T lymphoma cells (1) . These carbohydrate antigens are relevant markers for cancer diagnostic and prognosis, but they also represent potent targets for antitumor immune responses (2) . Targeting immune responses to such truncated variants of glycan chains expressed by tumor cells represents an important goal for the development of antigen-specific therapeutic vaccines against cancers (3) . Recent advances in the total synthesis of oligosaccharides expressed by tumor cells (4 , 5) open new possibilities for the development of synthetic carbohydrate-based vaccines. The implication of carbohydrate antigens in the metastatic process of tumor cells also makes these antigens relevant targets for the prevention of metastasis and recurrence of cancers (6) . Active antitumor immunization with an immunogen bearing the carbohydrate tumor markers may represent an alternative to conventional cancer therapy.

For immunization purposes, carbohydrates are traditionally conjugated to a carrier protein. Although this approach has proved to be successful (2 , 7 , 8) , it has major limitations, as follows: (a) hapten-carrier systems can be subjected to carrier-induced suppression of the immune response directed against the haptenic molecule (9 , 10) ; (b) the low molecular excess of the antigen over the carrier results in a small level of the desired antibodies compared to the total amount of antibodies produced; and (c) the protein conjugates present ambiguity in both composition and structure, which is a major obstacle for reproducible preparations. For these reasons, a fully synthetic immunogen, without protein carrier, could be of great interest in the development of a carbohydrate-based vaccine. Here, we tested the potential use of MAGs3 (11) as synthetic alternative immunogens. This system presents a high density of the carbohydrate antigen at the surface of a minor oligomeric inert lysine core. As a result, the immune response only focuses on the tumor antigen, thus limiting irrelevant antibody production. Moreover, such synthetic conjugates are particularly attractive for both their purity and accurate chemical definition. These features are, indeed, essential for quality control and consistent batch-to-batch vaccine production. The glycopeptide derivatives are synthesized by well-known standard solid-phase peptide synthesis methodology, which can be easily automated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Syntheses.
The Tn antigens ({alpha}-GalNAc-Ser/Thr) were synthesized by classical methods (12 , 13) . Syntheses of the MAG:Tn-PV, MAP:PV, Tn-PV, and PV were performed by the solid-phase methodology using the FMOC chemistry, as described previously (11) . After attachment of the ß-alanyl spacer to the Wang resin, the lysine core was assembled by coupling successively two levels of FMOC-Lys-(FMOC)OH, providing four amino groups. The lysine core was further elongated by the protected amino acids of the T-epitope sequence of the poliovirus (KLFAVWKITYKDT) to produce the MAP:PV. Ultimately, the {alpha}-GalNAc-Ser was incorporated to the four branches peptide which gave the MAG:Tn-PV construct after deprotection and cleavage from the resin, as reported previously (11) . All of the final constructs were purified by reverse-phase high-performance liquid chromatography and were characterized by amino acid analysis and electrospray mass spectrometry. The Tn3-TT glycopeptide [Ser({alpha}-GalNAc)-Thr({alpha}-GalNAc)-Thr({alpha}-GalNAc)-QYIKANSKFIGITEL] was prepared by incorporation, step by step, of the appropriate peracetyl-glycosylated FMOC-Ser/Thr in the peptide sequence using 2-(1H-Benzotriazole-1-yP)-1,1,3,3 tetramethyluronium tetrafluoroborate/N-Hydroxybenzotriazole (TBTU/HOBT) as the coupling reagent. Deacetylation of the sugar residue of the glycopeptide was achieved with a catalytic amount of sodium methoxide in methanol at pH 11. The crude product was purified by high-performance liquid chromatography (11) with a gradient from 10 to 35% and 14.74-min retention time. Electrospray mass spectrometry: 2623 (calculated, 2623.56). Amino acid analysis: Ala, 1 (1); Asp, 1.04 (1); Glu, 2.16 (2); Gly, 1.08 (1); Ile, 2.95 (3); Leu, 1.1 (1); Lys, 2.04 (2); Phe, 1.01 (1); Ser, 1.86 (2); Thr, 2.76 (3); and Tyr, 0.97 (1).

T-Cell Stimulation.
The recognition of the poliovirus T-cell epitope contained in the different constructs was analyzed using a specific T-cell hybridoma and A20 cells as antigen-presenting cells, as described previously (14) . T-cell hybridomas (105) were cultured with 105 A20 cells in the presence of the indicated construct in RPMI 1640 supplemented with 10% FCS, antibiotics, L-glutamine, and mercaptoethanol. Interleukin 2 synthesis following recognition by the T-cell receptor of hybridoma T cells was assessed by the proliferation of the interleukin 2-dependent CTLL cell line using [3H]thymidine.

Mice and Reagents.
Six- to 8-week-old BALB/c, SJL/J, and DBA/2 mice were from Iffa Credo. DBA/1 mice were from the animal colony of the Pasteur Institute. The anti-Tn mAb MLS128 (15) was provided by Dr. H. Nakada (Kyoto Sangyo University, Japan). Tn was conjugated to chicken OVA (Tn-OVA) at an initial molar ratio of 4000:1 using glutaraldehyde, as described previously (11) .

ELISA and Flow Cytometry.
Mouse sera were tested for anti-Tn antibodies by ELISA using the synthetic glycopeptide Tn3-TT or the parent peptide TT (YIKANSKIGITEL). Ninety-six-well microtiter plates (Nunc, Roskilde, Denmark) were coated with 0.1 µg of antigen per well in 50 mM carbonate buffer (pH 9.6) and incubated for 1 h at 37°C. After washing with PBS containing 0.1% Tween 20, the serially diluted sera in buffer (PBS plus 0.1% Tween 20–1% BSA) were added to the wells for 1 h at 37°C. Following three washes, wells were treated 1 h at 37°C using goat antimouse IgG or IgM peroxidase conjugate (Sigma Chemical Co., St. Louis, MO) and O-phenylenediamine-H2O2 was then added as substrate. Plates were read photometrically at A492 nm in an ELISA auto-reader (Dynatech, Marnes la Coquette, France). The negative control consisted of naive mouse sera diluted 100-fold. ELISA antibody titers were determined by linear regression analysis plotting dilution versusA492 nm. The titers were calculated to be the log10 highest dilution, which gave twice the absorbance of normal mouse sera diluted 1:100. Titers were given as the arithmetic mean ± SD of the log10 titers.

Mouse sera were also tested at a 1:250 dilution by flow cytometry on Tn-expressing human tumor cell lines, Jurkat and LS180, and on the TA3/Ha murine cell line. Cells were first incubated for 30 min with sera at 4°C in PBS containing 5% FCS and 0.1% sodium azide and then with an antimouse IgM/IgG goat antibody conjugated to FITC (Sigma). One % paraformaldehyde-fixed cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Antitumor Immunotherapy.
The murine mammary adenocarcinoma cell line, TA3/Ha was grown by passage on BALB/c mice. After i.p. administration of 1000 TA3/Ha cells, 6-week-old BALB/c mice were injected s.c. with 50 µg of MAG:Tn-PV or control MAP:PV construct with 1 mg of alum. Survival of treated and untreated mice was followed for 50 days. Statistical analysis of survival curves was performed with the Statview software (Abacus Concepts) using the log-rank test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Tn Antigen and the T-Cell Epitope Included into the MAG Retain Their Antigenicity.
As shown in Fig. 1Citation , the MAG we synthesized is composed of a dendrimeric lysine core structure with four arms. Each arm is linked to a CD4+ T cell epitope (PV peptide: KLFAVWKITYKDT sequence from the poliovirus type 1; Ref. 14 ) with a single {alpha}-N-acetylgalactosamine-serine residue (Tn) at the NH2 terminus {MAG:Tn-PV: [Ser({alpha}-GalNAc)]4-(PV)4-K2-K-ßAla; Ref. 11 }.



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  		Fig. 1. Schematic representation of the MAG:Tn-PV molecule.

 
The B-cell antigenicity of the Tn antigen included in the MAG:Tn-PV construct was analyzed using the MLS128 anti-Tn mAb, which was generated after immunization with the LS180 adenocarcinoma cell line derived from a colon cancer patient (15) . Fig. 2aCitation shows that the MLS128 mAb recognized the MAG:Tn-PV construct but not the parent construct devoid of the Tn antigen, MAP:PV [(PV)4-K2-K-ßAla]. This recognition was in the same range as that observed not only with Tn conjugated to the chicken OVA protein (Fig. 2a)Citation but also with the a-OSM (11) .



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  		Fig. 2. B- and T-cell antigenicity of the MAG:Tn-PV. a, the MLS128 mAb specific for Tn was tested by ELISA for reactivity to Tn-OVA conjugate ({blacktriangleup}), control MAP:PV ({circ}), and MAG:Tn-PV (•) coated at 1 µg/ml. b, stimulation of a PV-specific T-cell hybridoma by the PV ({square}), Tn-PV ({blacksquare}), MAP:PV ({circ}), and MAG:Tn-PV (•) constructs in the presence of A20 cells.

 
The poliovirus-derived PV peptide was included in the MAG:Tn-PV to elicit a T cell-dependent antibody response. However, linkage of carbohydrates to peptides can interfere with peptide binding to MHC-encoded molecules and with T-cell recognition (16) . For instance, Jensen et al. (17) showed that a T-cell hybridoma specific for a hemoglobin-derived peptide, glycosylated with {alpha}-GalNAc at a Thr residue, did not recognize the unglycosylated peptide in vitro. However, in this particular case, the glycosylation was introduced into the T-cell core sequence, whereas in our case, the Tn antigen was attached at the NH2 terminus of the PV peptide (KLFAVWKITYKDT), clearly distant from the core sequence, which was characterized as FAVWKITYKD (14) . As seen in Fig. 2bCitation , a PV-specific T-cell hybridoma recognize the unglycosylated PV peptide as well as the glycosylated Tn-PV peptide showing that the Tn saccharidic epitope did not alter the T-cell recognition of the PV peptide sequence. To analyze the T-cell antigenicity of the MAG:Tn-PV, A20 antigen-presenting cells were incubated with the MAG:Tn-PV or the control constructs in the presence of a PV-specific T-cell hybridoma (Fig. 2b)Citation . It is noteworthy that the presence of the Tn antigen dramatically increased the presentation of the PV peptide by MHC molecules because T-cell stimulation was achieved with a 10,000-fold less MAG:Tn-PV dose compared to the MAP:PV construct and to the PV free peptide (Fig. 2b)Citation . This effect was only observed with the dendrimeric MAG structure because the stimulation induced by the Tn-PV glycopeptide [Ser({alpha}-GalNAc)-PV] or the PV peptide was similar. The mechanisms underlying this enhancing effect remain unclear. However, because the formation of stable peptide/MHC complexes requires intracellular peptide loading on MHC molecules, the enhanced PV peptide presentation observed with the MAG:Tn-PV may stem from the intracellular processing of this construct.4 This result could also suggest that the MAG:Tn-PV endocytosis by antigen-presenting cells is mediated by the cross-linking of a GalNAc-specific receptor. After immunization of mice with the MAG:Tn-PV, T-cell responses specific for the PV epitope were stimulated in vivo (data not shown). Altogether, these results demonstrate that the Tn antigen on the MAG:Tn-PV construct is available for antibody binding and strongly enhances MHC presentation of the T-cell epitope, illustrating the potency of the MAG:Tn-PV construct to induce T cell-dependent anti-Tn antibodies.

The MAG:Tn-PV Induces High Titers of Anti-Tn Antibodies That Recognize Tn-positive Tumor Cell Lines.
The PV peptide contains a promiscuous MHC binding sequence, which enables its presentation to T cells by I-Ed and I-As MHC molecules (14) . Therefore, the immunogenicity of the MAG:Tn-PV was tested in different mouse strains expressing one of these MHC molecules. BALB/c (I-Ed), DBA/2 (I-Ed), and SJL/J (I-As) mice were immunized with the MAG:Tn-PV or with the control MAP:PV in alum, and sera were tested for anti-Tn antibodies (Table 1)Citation . The MLS128 mAb was shown to recognize three consecutive Tn antigens ([{alpha}-GalNAc]-Thr[{alpha}-GalNAc]) on a-OSM and glycophorin (18 , 19) . We, therefore, synthesized a glycopeptide, Tn3-TT, irrelevant to the MAG:Tn-PV amino acid sequence containing these three Tn antigens at the NH2 terminus of a linear peptide (TT) to evaluate by ELISA the level of anti-Tn antibodies. Immunization with the MAG:Tn-PV but not with the control MAP:PV induced anti-Tn IgG antibodies (mainly IgG1) in all three mouse strains tested. After three immunizations, Tn-specific IgM antibodies were still detected in BALB/c and SJL/J mice (Table 1)Citation . The Tn specificity of the antibodies using the Tn3-TT glycopeptide was assessed by the lack of recognition by all mouse sera of the parent TT peptide devoid of the Tn antigen. DBA/1 (I-Aq) mice, which do not respond to the PV peptide (14) , did not develop any anti-Tn antibodies following MAG:Tn-PV immunization, showing the T-cell dependency of the anti-Tn antibody response.


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  		Table 1 The MAG:Tn-PV-induced anti-Tn specific antibodiesa

 
To ensure that mouse sera were able to recognize the native Tn antigen, we analyzed the binding of these sera to tumor cell lines expressing Tn. {alpha}-GalNAc-Ser/Thr is present on glycoproteins expressed by the human Jurkat T-lymphoma cell line (20) and LS180 adenocarcinoma cell line (15) . Fig. 3, a and bCitation , shows that anti-Tn positive sera from BALB/c mice primed with the MAG:Tn-PV bound both human cell lines as efficiently as the MLS128 mAb, whereas sera from naive mice or MAP:PV immunized mice did not recognize these cell lines. These results demonstrate that anti-Tn antibodies induced by the MAG:Tn-PV recognize the native form of Tn on human tumor cells.



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  		Fig. 3. Recognition of tumor cell lines bearing the Tn antigen by sera from MAG:Tn-PV-primed mice. Flow cytometry analysis was carried out on human Jurkat (a) and LS180 (b) cells and (c) murine TA3/Ha cells incubated with sera (diluted at 1:250) collected from BALB/c: naive mice (· · · · ·), MAP:PV-primed mice (- - - - -), MAG:Tn-PV primed mice ({blacksquare}{blacksquare}) or the MLS128 mAb ( ). Binding was detected using FITC labeled antibodies specific for mouse immunoglobulin. The positive staining observed with the serum from the MAG:Tn-PV primed mouse is representative of five individually tested sera.

 
Immunotherapeutic Treatments with the MAG:Tn-PV Increase Mouse Survival and Resistance to Tumor Challenge.
A murine model has been developed to test active specific immunization against mucin-type carbohydrates using the TA3/Ha adenocarcinoma cell line (21) . In this model, treatment with bovine- or ovine-desialylated mucin prior and after the tumor challenge afforded partial protection (22) . Likewise, active immunotherapy in TA3/Ha-bearing mice using the T antigen ß-Gal(1–3){alpha}-GalNAc conjugated to the KLH protein together with cyclophosphamide was also able to strongly enhance mice survival (21) . FACS analysis of the TA3/Ha cell line (which expresses the Tn antigen on mucin epiglycanin; Ref. 23 ) indicated that this cell line was recognized by MAG:Tn-PV induced antibodies (Fig. 3c)Citation . Therefore, we grafted 1000 TA3/Ha cells into BALB/c mice and followed their survival after active anti-Tn specific immunotherapy using the MAG:Tn-PV (Fig. 4)Citation . Following TA3/Ha graft, control groups of mice that were either left untreated (group 1) or treated with MAP:PV (group 2) displayed average survival times of 25 and 24 days, respectively, whereas after treatment with MAG:Tn-PV (group 3), the average survival time was delayed to 30 days for mice that did not reject the tumor. The TA3/Ha cell is originated from A mouse strain and was shown to grow on many allogeneic mouse strains, but its malignancy slightly varies from one mouse strain to an other, depending on the genetic background (24) . Untreated BALB/c mice displayed a 10–20% rejection rate of the TA3/Ha after inoculation of 103 cells showing that the graft of TA3/Ha was a little less efficient in BALB/c than in other mouse strains used in studies performed with the same tumor model (21 , 22) . However, statistically significant differences were observed in resistance or survival of mice following TA3/Ha implantation between group 3 (48%) and the control groups 1 (22%) and 2 (19%; P < 0.02). These data show that the anti-Tn immune response induced by the MAG:Tn-PV increases the survival of tumor-bearing mice by rejecting the tumor graft expressing the Tn carbohydrate antigen.



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  		Fig. 4. Active specific immunotherapy in tumor-bearing mice. After i.p. administration of 1000 TA3/Ha adenocarcinoma cells (day 0), 6-week-old BALB/c mice were left untreated (group 1) or received, on days 2, 5, 10, and 17, a 50-µg dose of the MAP:PV (group 2) or the MAG:Tn-PV (group 3) mixed with 1 mg of alum, and then mice were monitored for survival. Cumulative results of three independent experiments are presented corresponding to 18 mice in group 1 and 21 mice in groups 2 and 3. Differences are statistically significant between groups 1 and 3 (P < 0.02) and between groups 2 and 3 (P < 0.01).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study represents the first successful attempt to induce a strong immune response to a carbohydrate tumor marker using fully synthetic carbohydrate-based constructs as immunogens. Indeed, we show that the MAG:Tn-PV injected to mice can induce a strong IgG antibody response specific for the carcinoma-associated Tn antigen, and we demonstrate that this antibody response is T cell dependent. A previous attempt by Toyokuni et al. (3) to develop such a synthetic immunogen was based on the Tn antigen linked via a peptidic arm to a palmytoyl backbone. However, in this study, Tn-lipopeptides mainly induced an IgM antibody response specific for Tn rather than an IgG response, and this antibody response was T cell independent due to the lack of T-cell epitope in these lipopeptides. In the context of tumor associated carbohydrate antigens, the induction of a T cell-dependent IgG antibody response represents a main goal because human sera already contain natural IgM antibodies (1) . In addition, it is striking to note that the presence of the Tn on the MAG:Tn-PV structure strongly enhances the level of MHC presentation of the PV T-cell epitope and probably contributes through enhanced T-cell activation to the high level of the IgG antibody response induced. The mechanism underlying this phenomenon is currently under investigation.

Our approach offers the advantages of a well-defined chemical structure and high purity, which are essential features for a safe vaccine, but it also provides a highly versatile peptidic core structure. Therefore, any carbohydrate moiety linked to an amino acid can be incorporated by standard solid-phase peptide synthesis methodology to always obtain the same carbohydrate content in the final compound. The glycopeptidic structure of the MAG construct may, thus, represent a future alternative to traditional protein conjugates carrying carcinoma or melanoma-associated carbohydrates that have been in clinical trials for several years (2 , 7 , 8 , 25 , 26) . The MAG strategy may also represent an efficient way to induce antibodies specific for bacterial oligosaccharides because the induction of a T-cell dependent IgG response is also important to achieve, for instance, in the case of Pneumococcus vaccination in infants (27) . Here, we did not compare the efficiency of our synthetic MAG construct to a carbohydrate-protein conjugate due to the paucity of qualitative and quantitative information on the latter. Moreover, such comparison remains difficult to achieve because the carbohydrate density on a carrier protein highly varies from one carrier protein to another and depends on many parameters, such as the conjugation method, the carbohydrate moiety, and the spacer arm that are used.

Our results demonstrate the potency of fully synthetic MAGs for active specific immunization, allowing, to a certain degree, the rejection of an implanted tumor-bearing aberrant glycosylations. Several hypothesis may explain the relative efficiency of antitumor immunity afforded by the MAG:Tn-PV treatment.

(a) On the basis of previous studies, total protection was never achieved in the TA3/Ha tumor model, and it seems that the immune mechanisms (humoral versus cellular response) that can lead to an efficient rejection of the TA3/Ha adenocarcinoma remain unclear (21 , 22) . Using a-OSM which expresses the Tn antigen, Singhal et al. (22) obtained 50% survival of TA3/Ha-bearing mice. In this study, immunization with a-OSM induced both an anti-Tn antibody response and an a-OSM-specific T-cell response. On the basis of putative sequence homology between OSM and TA3/Ha epiglycanin, these authors postulated that the T-cell response to glycopeptides carrying the Tn antigen could play a major role in the TA3/Ha rejection. Because the peptidic sequence of the MAG:Tn-PV is irrelevant to mucin-type protein sequences, clearly, MAG-treated mice did not benefit of such a T-cell response. Several studies demonstrated the ability of MHC molecules to bind synthetic glycopeptides to stimulate T-cell responses that are specific for the saccharidic moiety (16 , 17 , 28) . But it remains to be established that the MHC presentation of glycopeptides naturally occurs following the intracellular processing of glycoproteins.

Fung et al. (21) using the T antigen conjugated to KLH mixed with RIBI and in combination with a cyclophosphamide treatment obtained a higher protection level in the TA3/Ha tumor model than we did in this study. However, it should be noticed that the Tn antigen is the precursor of the T antigen, and it can be suggested that upon deglycosylation both T and Tn antigens are available on KLH as a target for the immune system. This phenomenon could contribute to the efficiency of this conjugate. Moreover, the cyclophosphamide treatment plays a key role in the efficacy of the T-KLH conjugate-based immunotherapy and was not used in our experiments.

(b) The affinity and the diversity of Tn-specific antibody response induced by the MAG:Tn-PV molecule is not probably as high as it should be to allow a total clearance of the TA3/Ha tumor in mice. Indeed, the Tn antigen is clustered along the mucin protein sequence but not in the MAG:Tn-PV molecule. Indeed, the MLS128 mAb was shown to recognize a three consecutive Tn epitopes on a-OSM and glycophorin (18 , 19) . Because the linear Tn-PV glycopeptide with a single Tn antigen was not recognized by the MLS128 mAb (11) and did not induce any Tn antibodies (data not shown), the binding of the MLS128 mAb to the MAG:Tn-PV is probably due to the flexibility of the peptidic arms rebuilding a Tn cluster. Therefore, we can expect that a MAG carrying several Tn antigen in each arm will be more closely related to the multimeric Tn motif displayed by mucin proteins. It also should be mentioned that multimerization of the Tn or the sialyl-Tn was shown to improve its immunogenicity when linked to a carrier protein (29 , 30) . Moreover, it was recently suggested for the sialyl-Tn antigen that carbohydrate clusters appear upon transformation of normal colonic tissues to malignancies (31) . Therefore, it seems clear that the configuration of the carbohydrate epitope is at least as important as the total carbohydrate content to induce an optimal antitumor response.

Tested in clinical trials for many years now, classical carbohydrate protein conjugates are still under study to optimize their immunogenicity (32) . The MAG molecule we tested here shows a high potency, but it only represents the first step of a new approach that needs to be much further developed, in particular by adding clustered carbohydrate epitopes, as mentioned above. Moreover, such immunogens probably would not focus on a single carbohydrate antigen but would, rather, combine various carbohydrate targets.

The use of a given Th cell epitope in conjunction with carbohydrates is a prerequisite for eliciting strong antibody responses, but this may limit the efficacy of the MAG immunogens considering the MHC polymorphism observed in the human population. To avoid this drawback, MAG structures have to include several T-cell epitopes with a particular focus on promiscuous MHC binding sequences, such as those described for tetanus toxin (33 , 34) , for which human individuals are already primed (35) . Because dendrimeric peptide structures seem to be more efficient compared to linear peptide sequences to induce CTL responses (36) , integration of CTL epitopes into MAG structures, such as MUC-1-derived peptides (37) for epithelial cancers, can also be achieved to widen the spectrum of the antitumor immune response. All these goals can be easily reached considering the versatility of the MAG system and can lead to a multicomponent therapeutic vaccine. Finally, it should be noticed that we have privileged the use of a mild adjuvant, alum, which is authorized in healthy human populations, showing that strong adjuvants are not required to induce anticarbohydrate specific immune responses by the MAG strategy. This latter point may be of major importance in extending the use of this strategy to bacterial oligosaccharides (27) for vaccinating a healthy population.


   ACKNOWLEDGMENTS
 
We are very grateful to Dr. H. Nakada for providing the MLS128 mAb and the LS180 cell line. We also thank Dr. F. Michel (Paris, France) for the Jurkat cell line and Dr. E. Roos (Netherlands Cancer Institute, Amsterdam, the Netherlands) for the TA3/Ha cell line.


   FOOTNOTES
 
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 work was supported by a grant from ARC. Back

2 To whom requests for reprints should be addressed, at Unité de Biologie des Régulations Immunitaires, 25–28 rue du Dr. Roux, 75724 Paris, Cedex 15, France. Phone: 33 1 45 68 83 52; Fax: 33 1 45 68 85 40; E-mail: rloman{at}pasteur.fr Back

3 The abbreviations used are: MAG, multiple antigenic glycopeptide; MAP, multiple antigenic peptide; FMOC, N-(9-fluorenyl)methoxycarbonyl; mAb, monoclonal antibody; OVA, ovalbumin; a-OSM, asialo ovine submaxillary mucin; KLH, keyhole limpet hemocyanin. Back

4 R. Lo-Man, unpublished data. Back

Received 11/10/98. Accepted 2/ 2/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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