Special Feature: Glycobiology Of Xenotransplantation And Cancer Part I

Immunology and Cell Biology (2005) 83, 440–448; doi:10.1111/j.1440-1711.2005.01347.x

Tumor-associated carbohydrate antigens: A possible avenue for cancer prevention

Yanfei Xu1, Alessandro Sette3, John Sidney3, Sandra J Gendler4 and Alessandra Franco1,2

  1. 1 Torrey Pines Institute for Molecular Studies, San Diego, California, USA
  2. 2 University of California San Diego, California, USA
  3. 3 La Jolla Institute for Allergy and Immunology, San DiegoCalifornia, USA
  4. 4 Mayo Clinic College of Medicine, Scottsdale, Arizona, USA

Correspondence: Associate Adj. Professor Alessandra Franco MD/PhD, University of California San Diego, La Jolla, CA 92093-0731, USA. Email: afranco@ucsd.edu

Received 10 February 2005; Accepted 13 February 2005.



Here we examine the use of glycopeptides containing tumour-associated carbohydrate antigens (TACA) as potential preventive vaccines for carcinomas. Our recent results suggest that CD8+ T cells (CTL) are capable of recognizing TACA in a conventional class I MHC-restricted fashion. The Thomsen–Friedenreich antigen (TF), a disaccharide, and Tn, its immediate precursor, are TACA largely expressed in carcinomas. TF and Tn can be successfully used as Th-independent vaccines when conjugated to designer peptides with optimal binding affinity for class I MHC molecules. TF- and Tn-specific CTL generated using this strategy are capable of recognizing TACA-expressing tumours in vitro, suggesting that glycopeptides are as effectively presented by class I MHC molecules as non-glycosylated peptides. Because the exact sequences of endogenously synthesized glycopeptides are unknown, the TACA-specific T cell repertoire elicited by carbohydrate-based vaccines is assumed to be degenerate. Here we report that mice genetically manipulated to develop TACA-expressing mammary tumours are not tolerant to glycopeptide vaccination. Moreover, we tested the immunogenicity of designer glycopeptides capable of binding multiple HLA alleles as a novel approach for the development of vaccines potentially useful for vaccination of a large fraction of the general population. Our results have suggested that CTL derived from normal donors respond with high efficiency to glycopeptides in vitro, opening a new avenue for the design of prospective vaccines for cancer prevention.


glycopeptide, immunotherapy, TACA, Tn.



Ehrlich suggested that the immune system 'recognizes' primary developing tumours prior to the discovery of cell types, molecules and effector mechanisms that represent the modern science of immunology1. It was not until 50 years later that the concept of 'cancer immunosurveillance' was proposed2, 3, which described lymphocytes as sentinels that recognize and eliminate continuously arising nascent transformed cells to maintain tissue homeostasis. Today we believe that the immune system controls, but also facilitates, tumour progression by 'sculpting' the immunogenic phenotype of developing tumours. This process has been defined as 'immunoediting' (reviewed in Dunn et al.4).

The lymphoid cells of acquired immunity play a critical and defined role in the host defence against cancer. In fact, B and T cells have been formally proven to be crucial in cancer surveillance, because mice deficient in the recombination activating gene (RAG-2-deficient) develop spontaneous malignancies5, and tumours grown in RAG-2-deficient hosts are less susceptible to immunoediting6.

CD8+ T cells (CTL) recognize MHC class I-restricted antigens derived from newly synthesized viral or neoplastic proteins and glycoproteins as a product of an endogenous processing pathway7. MHC class I molecules are constitutively expressed on somatic cells including epithelial cells. MHC class II molecules, however, are expressed on lymphoid and myeloid cells, but rarely on somatic cells, and are only induced by inflammatory stimuli, which is the reason why CTL have been preferentially targeted in cancer immunotherapy.

Much time has been expended in an effort to characterize the tumour epitopes recognized by T cells8, using diverse technologies9. Relevant tumour-specific antigens have been successfully discovered in some tumour types, including melanoma9 and breast cancer10, 11, 12, 13, 14, 15. Unfortunately, tumour antigens are often tissue-specific and do not share antigenic patterns, meaning that it is very difficult to generate preventive vaccines for a large population.

A group of tumour-associated antigens have been identified and characterized by virtue of their reactivity with antibodies and lectins that are carbohydrate in nature and called tumour-associated carbohydrate antigens (TACA)16. Tumor associated carbohydrate antigens (TACA) are broadly expressed as a product of aberrant glycosylation in a large number of tumours and may represent a unique tool for prophylactic or therapeutic vaccination17, 18, 19, 20, 21, 22. Aberrant glycosylation has been found in all tumour cells examined and the expression of TACA, the result of this process, influences the prognosis and survival of cancer patients in a manner that is proportional to the degree of expression23. TACA are present in tumours more frequently than oncogene products (e.g. myc, rask, HER2/neu) and their association with tumour progression is stronger than the deletion or inactivation of tumour-suppressing genes (e.g. p53, p16)22. In fact, TACA are not only tumour markers, but constitute part of the machinery that is essential for inducing metastasis and invasiveness24.

The Thomsen–Friedenreich antigen (TF or T) (beta-Gal-[1-3]-alpha-GalNAc-O-serine)25, 26 and its precursor Tn (GalNAc-O-serine)27 are exclusively expressed in carcinomas but not in normal tissues28, 29. Their small size and broad expression during the early stages of cancer transformation in several tumour types including bladder, colorectal, gastrointestinal, prostate, ovarian and lung carcinomas30, 31, 32, 33, 34 indicate that TF and Tn are potential targets for CTL-based cancer prevention.

Our previous work demonstrated that it is possible to generate in vivo an anti-TF- and anti-Tn-specific CTL repertoire that is capable of recognizing TACA-expressing tumours, including MUC1-expressing tumours, by immunizing mice with designer glycopeptides35.

Here we show that mice that have been genetically manipulated to develop spontaneous mammary tumours that express the MUC1 glycoprotein36 are not tolerant to glycopeptide vaccination. This lack of tolerance is independent from age and tumour progression.

Importantly, conjugation with small carbohydrate molecules to the peptides does not affect binding to the MHC. Moreover, Tn-containing natural viral sequences capable of binding multiple class I MHC alleles are highly immunogenic in vitro when tested in normal human donors, indicating that TACA-based CTL vaccines hold great promise for cancer prevention.


Materials and methods

MUC1 transgenic mice and development of MUC1/ MT double transgenic mice

The MUC1 transgenic mice were developed by injection of a 10.5 kb genomic SacII fragment containing the entire MUC1 gene sequence, as well as 1.5 kb of 5 sequence and 800 bases of 3 sequence into the pronuclei of fertilized C57BL/6 mouse eggs, which were then transferred to pseudopregnant females37. Founder mice were identified by Southern blot analysis and one founder (79.24), which showed appropriate tissue-specific expression of MUC1, was selected for further study. The level and pattern of MUC1 expression in the various organs was similar to that seen in humans. Low levels of expression are observed in normal tissues, with overexpression occurring following the development of tumours in the mammary gland and pancreas38, 39.

Mice were genotyped by PCR using the following primers: 5'-CTTGCCAGCCATAGCACCAAG-3' (bp 745–765)40 and 5'-CTCCACGTCGTGGACATTGATG-3' (bp 1086–1065)37, 40. MUC1 transgenic mice were maintained continuously on the C57BL/6 background.

Development of spontaneous tumours in MUC1 transgenic mice

To generate spontaneous tumours, MUC1 transgenic mice were bred with mice carrying the MMTV-driven polyoma middle T antigen (MT) to create MUC1/MT mice39, 41. Prior to generating the double transgenics, the MT mice had been backcrossed 10 generations onto C57BL/6 mice, which made them congenic. The offspring were screened for the polyoma middle T transgene using PCR on 100–500 ng genomic DNA prepared from tail snips with the following primers: 5'-AGTCACTGCTACTGCACCCAG-3' (bp 282–302) and 5'-CTCTCCTCAGTTCTTCGCTCC-3' (bp 817–837)39. PCR reactions (50 (L) contained 2% deionized formamide, 0.2 mmol/L dNTP mix, 1times PCR buffer, 1 (mol/L forward and reverse primers, and 1.75 mmol/L MgCl2. PCR conditions were as follows: a 10 min hot start at 94°C and 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, followed by 72°C for 10 min. Polyoma middle T antigen-positive female mice developed multifocal mammary adenocarcinomas. Tumours were excised when they reached 1 cm in diameter, then they were fixed in methacarn, embedded in paraffin and stained for MUC1 using a humanized HMFG-1 mAb (at 1:5000) and a secondary peroxidase-conjugated antihuman IgG (1:50). This method enabled better differentiation between low and high levels of MUC1 expression while avoiding the background staining that was detected when using an unlabelled murine mAb.

Peptides and glycopeptides

The peptides used in this study were synthesized by Fmoc chemistry using a multiple peptide synthesizer (Symphony/Multiplex, Protein Technologies, Tucson, AZ, USA). Peptides were cleaved automatically on the synthesizer using trifluoroacetic acid as a cleavage reagent. Peptides were greater than or equal to97% pure as assessed by C18 reverse phase HPLC, and the identity of the peptides was verified by mass spectroscopy. One designer alanine-rich peptide with the appropriate Kb anchors, and two natural sequences derived from the hepatitis B virus (HBV) and one from HIV were Tn-conjugated either in position 4 or in position 5 to produce the Kb-binding designer alanine-rich AIIA(GalNAc-O-S)FAAL, HBV pol 635–643 GLYS(GalNAc-O-S)STVPV, HBV core 18–27 FLP(GalNAc-O-S)DFFPSV, and HIV gp 120 272–281 TLT(GalNAc-O-S)CNTSV.

The Tn-containing glycopeptides (Tn (GalNAc-O-)) utilized in this study were prepared by solid phase synthesis using glycosylated amino acids as building blocks, as previously described42. Glycopeptides were greater than or equal to97% pure as assessed by C18 reverse phase HPLC, and the identity of the peptides was verified by mass spectroscopy.

MHC binding assay

The EBV-transformed cell lines JY (A*0201, B*0702), M7B (A*0202/A*0301, B*3501/B*5301), FUN (A*0203/A*0301, B*4601/B*3501), CLA (A*0206/A*2402, B*0801/B*3502), and AMAI (A*6802, B*5301) were used as the primary sources of HLA A class I molecules. Cells were maintained in vitro and HLA molecules were purified by affinity chromatography as previously described43. Quantitative assays to measure the binding of peptides to HLA A*0201, A*0202, A*0203, A*0206, and A*6802 molecules were based on the inhibition of binding of a radiolabelled standard peptide43, 44. Briefly, 1–10 nmol/L of radiolabelled peptide was coincubated at room temperature with 1 (mol/L to 1 nmol/L of purified MHC in the presence of 1 (mol/L human beta2-microglobulin (Scripps Laboratories, San Diego, CA, USA) and a cocktail of protease inhibitors. After a 2 day incubation, binding of the radiolabelled peptide to the corresponding MHC class I molecule was determined by capturing the MHC– peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-one, Longwood, FL, USA) coated with the W6/32 antibody, and measuring bound cells using the TopCount microscintillation counter. In addition, following the 2 day incubation, the percentage of MHC-bound radioactivity was determined by size exclusion gel filtration chromatography using a TSK 2000 column.

Immunization protocols and characterization of CTL lines and clones derived from MUC1/MT double transgenic mice

Glycopeptides were emulsified in IFA and injected subcutaneously at a concentration of 50 microg, together with 140 microg of an IAb-restricted Th epitope, the hepatitis B virus core antigen (HBc) sequence 128–140 (TPPAYRPPNAPIL)45. Seven days after priming, mice were killed. Splenocytes from each experimental group were pooled and stimulated in vitro in the presence of the glycopeptide that was used as an immunogen and, as an APC source, irradiated syngeneic B cell blasts activated in vitro for 48 h by culturing splenocytes with LPS (from Salmonella typhosa: Sigma, St Louis, MO, USA) and dextran sulphate (DxS; Pharmacia Biotech AB, Uppsala, Sweden). The culture medium consisted of RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) supplemented with 20 mmol/L glutamine, 100 microg streptomycin, 100 U/mL penicillin, 1 mM sodium pyruvate, 0.1 mmol/L non-essential amino acids (Life Technologies), 50 micromol/L 2-ME, and 10% heat inactivated FCS (Life Technologies). After 5 days in culture, cells were collected, purified over Ficoll gradients, and T cell blasts were cultured in complete RPMI conditioned with supernatant from splenocytes activated with ConA (Sigma, St Louis, MO, USA) as an IL-2 source. Two days later (day 7 in culture), T cells were tested for CTL activity. Carbohydrate and peptide specificity were studied in a standard 51Cr release assay using the lymphoma EL-4 (H-2b) cell line as targets at different E : T ratios in duplicate, with and without antigens. Net specific lysis was calculated by subtracting the cytolytic response to EL-4 cells in the absence of glycopeptides or peptides, which was defined as background. Data were calculated as percentage cytotoxicity = [(sample – spontaneous release)/(maximum release – spontaneous release)] times 100.

T cell clones were generated from selected Tn-specific CTL lines at 6–7 days following a single in vitro stimulation with the immunizing glycopeptide to avoid in vitro selection. Briefly, after Ficoll purification, T cell blasts were plated in 96 well U- bottom plates at 0.5 cells per well in the presence of 1 microg/mL glycopeptide and irradiated LPS/DxS activated B cell blasts. Growing wells were expanded with ConA supernatant and restimulated with irradiated B cell blasts and glycopeptides. CTL clones were tested for specificity by using EL-4 targets. Endogenous recognition of the carbohydrate expressed by tumour cells was determined by using as targets for the 51Cr release assay the mammary tumour line MMT, which is derived from a spontaneous syngeneic tumour in a MMT female (double transgenic MUC1/MT) and a MUC1-transfected B16 melanoma37. TACA expression on tumour cells was tested before the experiment in in vitro experiments by using anti-MUC1 monoclonal antibodies, the CT246 and anti-TF monoclonal antibodies, which were kindly provided by Dr Bo Jansson (Bioinvest, Luden, Sweden)47.


In this study, PBMC were derived from volunteer normal donors aged between 40 and 57. Donors were genotyped by PCR to determine their human HLA class I loci.

Human CTL cultures

Natural peptide sequence and corresponding Tn (GalNAc)-modified glycopeptides were compared side by side for their ability to prime in vitro naive cytotoxic T cells derived from PBMC of normal human donors by using autologous, antigen-pulsed, mature dendritic cells (DC) as APC.

Autologous DC were cultured with GM-CSF (kindly provided by Kirin, Gumna, Japan) and IL-4 (Peprotech, Rocky Hill, NJ, USA) and matured with 100 U/mL TNF-alpha as described previously48.



MUC1/MMT are not tolerant to glycopeptide vaccination

TF and Tn are expressed by mucins encoded in humans by the MUC1 gene35. MUC1 is a large glycoprotein that is aberrantly expressed in several carcinomas, which makes it an attractive target for immunotherapy49. Here we address CTL tolerance to TACA-containing glycopeptides in MUC1/MT mice that spontaneously develop MUC1-expressing mammary tumours within 14 weeks after birth39. These mice were exposed in the embryo to TF and Tn TACA antigens. TF and Tn are precursors of the MN blood group substance expressed on glycophorin50, which is also expressed on the MUC1 found on tumour cells35.

The experiments in MUC1/MT transgenic mice were carried out with the Tn-containing alanine-rich glycopeptide AIIA(GalNAc-O-S)FAAL, which has previously been described as more immunogenic than its TF-containing variant AIIA(beta-Gal-(1–3)-alpha-GalNAc-O-S)FAAL35. In these experiments, MUC1/MT mice were chosen according to age and stage of tumour development. Two mice per group (aged 14, 18, and 24 weeks) were immunized with the glycopeptide emulsified in IFA, coinjected subcutaneously together with an IAb-restricted Th epitope, the HBc sequence (TPPAYRPPNAPIL) in repeated experiments.

Seven days after priming, the mice were killed. Splenocytes from each experimental group were pooled and stimulated in vitro in the presence of the glycopeptide that was used as the immunogen and irradiated B cell blasts as APC. After 5 days in culture, cells were collected, purified in a Ficoll gradient, and T cell blasts were cultured in complete RPMI conditioned with supernatant from ConA-activated splenocytes as an IL-2 source. Two days later (day 7 in culture) T cells were tested for CTL activity. Carbohydrate and peptide specificity were studied in a standard 51Cr release assay using the lymphoma EL-4 (H-2b) cell line as a target at different E : T ratios in duplicate, with and without antigens.

Carbohydrate specificity was investigated by comparing the responses of the glycopeptide and its respective non-glycosylated counterpart. Net specific lysis was calculated by subtracting the cytolytic response to EL-4 cells alone without antigens, which was defined as background.

A strong CTL activity in response to the immunizing glycopeptide was generated in MUC1/MT transgenic mice, regardless of their age and tumour progression (Figure 1). In contrast, greatly diminished activity was observed against the same peptide in the non-glycosylated form, demonstrating the generation of Tn-specific CTL that either do not use TCR contacts within the peptide backbone or are not activated if such contacts exist, as previously shown in wild type mice35.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The CTL response to a Tn-containing glycopeptide vaccine in mice affected by Tn-expressing mammary tumours. The specificity of primary CTL cultures tested in a 51Cr release assay is shown. Labelled EL-4 cells (H-2b) were incubated with different numbers of T cell blasts in the presence of 1 microg/mL immunogen glycopeptide or unglycosylated peptide backbone. EL-4 cells plated with T cell blasts in the absence of antigens were assessed in duplicate at every E : T ratio to determine the extent of non-specific lysis. Cell culture supernatants were harvested 4 h later and the percentage of 51Cr released was calculated as (sample – spontaneous release)/(maximum release – spontaneous release) times#100. Background lysis has been subtracted and net lysis is shown. Empty symbols represent carbohydrate specificity, whereas filled symbols indicate peptide specificity. A representative experiment where two mice at each of the ages 14 weeks, 18 weeks and 24 weeks, which were carrying tumours of different sizes, were studied is shown.

Full figure and legend (22K)

Out of 30 MUC1/MT mice studied, 27 (90%) responded to Tn vaccination, suggesting that designer glycopeptides function as 'altered self'. In fact, no response to Tn has ever been detectable in the spleen of unprimed MUC1/MT mice in repeated experiments, proving that TACA-containing glycopeptides have a high potential to break the immunological tolerance to 'self' tumour antigens.

The immunotherapeutic potential of Tn-containing vaccines in this lineage are under investigation in our laboratory.

Tn-specific T cell clones kill MUC1-expressing tumours in vitro

The ability to recognize Tn-expressing tumour cells by using CTL generated from MUC1/MT mice immunized with glycopeptides is critical for the design of this vaccine. Tn-specific CTL clones were generated from independent T cell lines derived from MUC1/MT mice immunized with AIIA(GalNAc-O-S)FAAL. The T cell clonal repertoire after glycopeptide immunization was predominantly Tn-specific and had high avidity for the carbohydrate moiety, supporting the results obtained with primary T cell lines and confirming the observations obtained in wild type mice35.

Two tumour cell lines were selected as targets for these experiments: (i) MMT, a syngeneic mammary tumour carcinoma derived from a double transgenic female MUC1/MT mouse (MMT), that expresses Tn in the context of MUC1; and (ii) a MUC1-transfected B16 melanoma cell line37.

The dose–response relationship for the glycopeptide immunogen suggests the generation of high-affinity Tn-specific CTL clones (Figure 2a). Moreover, because a large percentage of the syngeneic mammary tumour cells and the melanoma cell line transfected with MUC1 were killed, it indicates that Tn-specific CTL can recognize the TACA endogenously expressed in a MHC class I-dependent fashion (Figure 2b).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Characterization of Tn-specific CTL clones. Dose–response relationship of five representative anti-Tn CTL clones to AIIA(GalNAc-O-S)FAAL measured in a classical 51Cr release assay at a 2:1 E : T ratio. In vitro killing of the syngeneic mammary tumour cell line MMT and the melanoma cells transfected with MUC1 and B16/MUC1, by anti-Tn specific CTL clones measured at a 20:1 E : T ratio. (filled square), 1A7; (), 3B9; (square with dots), 1B3; (square with 45 degree lines), 4E5; (square), 1C8.

Full figure and legend (29K)

Because the sequences of the endogenous glycopeptides that present Tn in the context of the tumour cell surface are unknown, the CTL clonal repertoire is shown to be highly degenerate. Differences in the magnitude of the CTL response against the two tumours by single clones and between clones prove that different glycopeptides are endogenously presented to T cells and that the Tn-specific T cell repertoire is diverse and heterogeneous.

Glycopeptides can be designed to bind multiple class I MHC alleles

To transfer the TACA-containing glycopeptide vaccine strategy from mice to humans, we designed Tn-containing glycopeptides that are potentially capable of binding multiple HLA class I alleles51. Class I MHC molecules are expressed at three loci in the human genome and comprise a large number of different molecules, with patterns of sequence conservation and polymorphism52. Each of the class I MHC proteins is capable of binding a set of peptides of diverse sequence motifs53. The mechanism by which this sequence degenerate binding occurs has been elucidated in the past 10 years54, 55, 56, 57, 58, 59. Interactions between conserved MHC side chains and polar main chain atoms of the peptide termini provide a universal peptide binding capability. Additional binding energy and limited peptide sequence are selectively introduced by the binding of a few peptide anchor side chains in pockets formed by the MHC side chain that are polymorphic53, 58, 60.

Crystal structures of murine and human class I MHC complexes54, 61 reveal that bound peptides are mainly expressed in the centre of the cleft, where the peptides 'bulge' from the floor of the peptide binding site.

Our experimental design takes advantage of this knowledge. We designed glycopeptides that are potentially capable of binding multiple class I MHC alleles, hoping to skew the fine specificity of the T cells towards the carbohydrate by introducing serine substitutions and the conjugation of O-linked Tn at major TCR amino acid residue(s). Two of the peptides that were glycosylated are well-known HBV-derived CTL epitopes previously described in the literature, including the immunodominant HBV core 18–2759, 62, 63, 64, 65, 66, and the HBV pol 635–643.66, 67 Another epitope chosen for glycosylation was the previously described HIV gp 120 272–28168, 69. Each of the peptides studied had a serine residue at a position predicted to be a major TCR contact and are not likely to interfere with HLA binding capacity, which is ideal for the attachment of sugar residues.

As shown in Table 1, peptides were confirmed to be good A*0201 binders, with binding affinities in the 0.2–72 nmol/L range. Each of the Tn-containing glycopeptide variants also bound with good affinity, and in general bound as well or better than the wild type peptide. The A*02 degenerate binding capacity of the glycopeptides was also retained.

These observations justify the use of the glycopeptide variants in functional experiments to evaluate their immunogenicity and also suggest that they are good candidates for the development of preventive or therapeutic vaccines with broad population coverage.

Tn-containing glycopeptides prime with high efficiency naive CD8+ T cells derived from normal A2 donors

The three glycopeptides described in Table 1 were studied for their ability to prime in vitro naive cytotoxic T cells derived from PBMC of normal donors. Seven HLA-typed A2 donors, aged between 40 and 57 years, were included in this study.

Autologous, antigen-pulsed, mature DC were used as APC for in vitro priming. Twelve CTL lines/glycopeptides and 12 CTL lines/peptides were established by coculturing in 48 well flat bottomed plates 5 times 105 fresh PBMC per well and 1 times 105 mature DC per well, which had previously been pulsed for 2 h with glycopeptides or peptides. Each well defined an individual CTL line.

Analysis using FACS to determine the number of CD8+ T cells in the PBMC population indicated that CD8+ T cells represented an average of 25% of the total PBMC, ranging from 13% to 37% in different donors. The small number of CD8+ T cells/T cell lines enabled us to determine the approximate precursor frequency of in vitro primed naive CTL.

Each experiment also included 12 CTL lines primed in vitro with an irrelevant Kb-restricted peptide derived from chicken ovalbumin (OVA) 57–6470 as a negative control. The T cell cultures received IL-2 (Peprotech) twice (day 5 and 8) and they were tested for specificity at day 10 in a classical 51Cr release assay.

T2 cells71 were used as targets in these experiments. Each T cell line was assayed at a 3:1 E : T ratio. All the T cell lines were tested individually for their ability to recognize the glycopeptide immunogens, the peptide backbones and the T2 cell line used as targets in the absence of antigens (background control).

The results of these experiments are summarized in Table 2, where the number of specific lines giving a net specific lysis greater than or equal to20% in seven normal donors is reported. Carbohydrate specificity was defined as a specific response to the glycopeptides minus the specific response to the corresponding peptide backbone.

A large number of glycopeptide-specific and peptide-specific CTL lines were derived with a single stimulation in vitro in the absence of T cell help, suggesting that antigens that bind HLA molecules with high affinity are Th-independent as previously shown in the Kb model35. Moreover, glycopeptides are more immunogenic than peptides, as shown by the number of specific T cell lines obtained by summarizing and comparing the specific CTL responses to Tn-modified and natural peptide sequences. The large number of specific CTL lines obtained, starting from a low number of CD8+ T cells per T cell line, strongly suggest a high precursor frequency of the T cell carbohydrate-specific repertoire in the periphery.



We previously demonstrated that TACA-containing designer glycopeptides could generate, in vivo, a CTL repertoire that is highly carbohydrate-specific and capable of killing TACA-expressing tumours in vitro 35. Here we show that the same vaccination strategy is successful in generating TACA-specific CTL in mice that develop spontaneous mammary tumours that express the TACA antigen in the context of mucin35, namely MUC1/MT39.

T cell clones derived from immunized MUC1/MT mice recognize the glycopeptide antigens with high affinity, and kill in vitro Tn-expressing tumour cells in the context of the MUC1 glycoprotein72, proving that this immunization strategy can break T cell tolerance to 'self' tumour antigens. In fact, Tn-specific CTL are not detectable in unprimed litter-mates.

In the present study, we found that TACA are presented in the context of endogenous glycopeptides of unknown sequences, showing that the CTL response is very degenerate, which is a clear advantage in immunotherapy73.

Because the glycosylated amino acid linker (serine) is also recognized by carbohydrate-specific TCR, we believe that glycosylation of suitable amino acid residues within MHC– peptide complexes may be very common in vivo, as suggested by the rabies virus model74. Similarly, as glycopeptides are presented to T cells in the thymus, they may contribute to positive selection, shaping the repertoire of T cells that reach the periphery. In fact, the large majority of TACA are embryonic antigens, and are not represented in normal tissues22.

Moreover, our results using A2-restricted Tn-conjugated viral sequences indicate that Tn-conjugation does not affect binding to HLA class I molecules and, in fact, increases the immunogenicity of wild type sequences. In support of this assertion is the observation that the TACA conjugation was well tolerated by TCR. The amino acid contacts used for glycosylation in the HBV core 18–2763, 75 and the HIV gp 120 272–281 determinants76 were shown to be susceptible to mutations due to immune pressure, leading to CTL inhibition. However, serine glycosylation at the same position did not negatively affect, but actually increased the immunogenicity compared with the wild type sequences.

The evidence that normal donors respond to glycopeptide vaccination in vitro is very promising for future clinical trials. We have also shown that glycopeptides containing TACA antigens expressed in different tumour types can be designed that bind multiple class I MHC alleles, indicating the possibility for preventive vaccination of a large fraction of the general population.



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The authors thank Dr Howard M. Grey (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA), who was a pioneer in the field of T cell recognition of carbohydrates. This work has been supported by a National Institute of Health grant R29CA78657, a Department of Defense grant BC010002 and a Alzheimer's and Aging Research Center grant to A. Franco.


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