WO2004055183A2

WO2004055183A2 - Carcinoembryonic antigen-specific immunodominant epitope recognized by cd4+t cells and uses thereof

Info

Publication number: WO2004055183A2
Application number: PCT/IT2003/000791
Authority: WO
Inventors: Maria Pia Protti; Paolo Dellabona
Original assignee: Fondazione Centro San Raffaele Del Monte Tabor
Priority date: 2002-12-17
Filing date: 2003-12-01
Publication date: 2004-07-01
Also published as: AU2003295189A8; AU2003295189A1; WO2004055183A3

Abstract

CEA epitopes are specifically recognized by CD4+T cells and are useful also in helping the induction of CD8+ response and amplification of memory effect.

Description

CARCINOEMBRYONIC ANTIGEN-SPECIFIC IMMUNODOMINANT EPITOPE RECOGNIZED BY CD4+ T CELLS AND USES THEREOF

TECHNICAL BACKGROUND CD4+ T cells are implicated in fundamental functions in the induction of productive antitumor immunity, comprising: i) help for CD8+ T cells activation through interaction with antigen presenting cells (APC), such as dendritic cells, displaying tumor peptides in association with both MHC class I and II, ii) maintenance of CD8+ T cell memory, in) indirect effector functions through activation of macrophages and eosinophils and TFN-a dependent anti-angiogenesis, and iv) direct recognition of MHC class II positive tumor cells (1-4).

The carcinoembryonic antigen (CEA) is a 180-Kda glycoprotein expressed at high levels in colon epithelial cells during embryonic development but at significantly lower levels in adult tissue (rev. in 5). It is overexpressed in almost all colorectal cancers, 70% of non-small-cell lung cancers, and approximately 50% of breast cancers (5). The CEA, or CD66e, belongs to the CD66 immunoglobulin (Ig) super- gene family that comprises also the CD66a, CD66b, CD66c, CD66d and CD66f molecules (6). Some of them, in addition to epithelial cells, are expressed in normal cells of hematopoietic origin (i.e. leukocytes) and share with CEA regions of high homology (6).

The function of CEA in normal colon epithelial cells and in tumor cells is not entirely clear. It acts as an adhesion molecule with probably different functions in normal versus neoplastic colon tissue, due to different localization in the two tissues (7). The altered pattern of localization in tumor cells might help to disrupt intercellular adhesion, with disorganized growth and movement of malignant cells, pointing to a role in the development of the metastatic disease. CEA has been shown also to inhibit cell death (8), and to cooperate in cellular transformation with several proto- oncogenes, such as BCL2 and C-myc (9). Several reports have shown that CEA is immunogenic. Some groups found evidence of anti-CEA antibodies in colon and breast cancer patients (10-11). The potential to elicit T-cell responses was first suggested by the observation that individuals who had colon cancer often exhibited a delayed-type hypersensitivity response to purified CEA protein (12). More recently, recombinant vaccinia viruses expressing CEA were administered to cancer patients, and CEA-specific T cells were subsequently cloned from these patients, demonstrating that T cells can recognize CEA (13). A few CEA epitopes recognized by CD8+ T cells (14-21) and one recognized by CD4+ T cells have been identified so far (22).

The expression profile, its role in tumor progression and its immunogenicity make CEA an attractive target for immunotherapeutic purposes and worthwhile further efforts for T cell epitope identification.

Patent application WO 02/22803 describes the identification of CEA epitopes able to activate CD4+ T cells involved in the pathogenesis or protection from cancer diseases. The patent application discloses three peptides comprised into a 35 amino acid peptide region wherein no more than 30% of amino acid residues are substituted.

DESCRIPTION OF THE INVENTION

By using a combination of bio-informatics and cellular approaches, the authors have identified an immunodominant CEA epitope recognized by CD4+ T cells from healthy donors and colon cancer patients in association with 7 HLA-DR alleles. The authors show here that the identified sequence contains naturally processed epitope(s) and that CD4+ T cells specific for this sequence do not cross-react with analog sequences, present on the homologous CD66 proteins, and potentially presented by normal hematopoietic cells. The invention provides CEA epitopes that are specifically recognised by CD4+T cells to be used also in helping the induction of CD8+ response and amplification of memory effect. Moreover CEA specific CD4+ T cells may have direct or indirect (through macrophages or eosinophils) antitumor response.

The authors used a MHC class II epitope prediction algorithm (TEPITOPE) to select 11 sequence segments of CEA that could form promiscuous CD4+ T cell epitopes and used synthetic peptides corresponding to the predicted sequences to propagate in vitro CD4+ T cells from healthy donors and colon cancer patients. CD4+ T cells from all subjects strongly recognized the sequence segment (LWWVNNQSLPVSP), repeated at residues 177-189 and 355-367. Most importantly, the authors demonstrated that this highly immunodominant region contains naturally processed epitope(s). Cross-recognition experiments with peptide analogs present on the CD66 homologous proteins showed that CEA177-189/355-367 specific CD4+ T cells did not recognize the analogs demonstrating that recognition of the immunodominant epitope is CEA-specific. These data suggest that the repertoire of CEA177-189/355- 367 specific CD4+ T cells might have been shaped by a selective process to exclude CD4+ T cells specific for CD66 homologues expressed on leukocyte, while preserving the CEA specific repertoire. The features of strong immunogenicity and immunodominance in the absence of potential induction of autoimmunity make the identified CEA epitope of particular interest for the development of antitumor vaccines. Single amino acid substitutions of the identified region largely decrease or abrogate T cells proliferation and therefore the immunogenic activity. The expression profile, its role in tumor progression and its immunogenicity make CEA an attractive target for immunotherapeutic purposes and worthwhile further efforts for T cell epitope identification. The peptides of the current invention have been further validated in that T cells primed with these epitopes are able to recognize the native CEA protein after processing by autologous antigen presenting cells and by tumor cells expressing

CEA.

It is therefore an object of the invention an isolated CEA class II binding peptide able to react with CD4+ T cells having an amino acid sequence comprised in the sequences of peptides of the following list: SEQ ID. No.l, SEQ ID. No.2, SEQ ID. No.3, SEQ ID. No.4, SEQ ID. No.5, SEQ ID. No.6, SEQ ID. No.7, SEQ LD. No.8,

SEQ ID. No.9, SEQ ID. No.10 and SEQ ID. No.l 1. Preferably the sequences of peptides are the following list: SEQ ID. No.2, SEQ ID. No.3, SEQ ID. No.5, SEQ ID. No.7 and SEQ ID. No.11. More preferably the sequences of peptides are comprised in SEQ ID. No.3 or SEQ ID. No.5. It is another object of the invention an isolated nucleic acid encoding the peptide according to any of previous claims. It is another object of the invention an expression vector able to efficiently express the isolated nucleic acid, and a host cell transformed with the expression vector. The peptide of the invention is able to specifically activate CD4+ T cells if biund to MHC class TJ molecules. Therefore it is another object of the invention a multimer class II MHC complex comprising at least one peptide of the invention, preferably the multimer is a tetramer.

It is another object of the invention an antigen presenting cell expressing at least one of peptides of the invention.

It is another object of the invention a method for detecting CD4+ T cells reacting with at least one of peptides of claim 1 in a biological sample comprising the steps of:

- exposing the biological sample to a multimer class II MHC complex comprising at least one peptide of the invention in a condition allowing the binding of the multimer complex to cells; - detecting reacted cells by means of a revealing system.

Preferably the revealing system consists of a detectable molecule bound to the multimer class II MHC complex comprising at least one peptide of the invention.

Preferably the detectable molecule is a chromophore.

In an alternative embodiment, the revealing system consists in the detection of molecules produced by CD4+ T cells upon activation by the peptide of the invention by reacting with specific molecule reagents, as antibodies and revealing means.

Preferably the molecules produced by CD4+ T cells upon activation by the peptide of the invention belong to the following group: performs, granzyme and cytokines, more preferably cytokines are gamma-IFN, IL-10, IL-4, IL-5. It is another object of the invention a method for isolating CD4+ T cells reacting with at least one of peptides of the invention in a biological sample comprising the steps of:

- exposing the biological sample to a multimer class II MHC complex comprising at least one peptide of the invention bound to a chromophore in a condition allowing the binding of the multimer complex to cells;

- separating reacted cells by FACS. It is an alternative embodiment of the invention a method for isolating CD4+ T cells reacting with at least one of peptides of the invention in a biological sample comprising the steps of:

- exposing the biological sample to a multimer class II MHC complex comprising at least one peptide of the invention in a condition allowing the binding of the multimer complex to cells;

- exposing the reacted sample to a solid phase-reagent specific for molecules produced by CD4+ T cells upon activation by the peptide of the invention;

- separating the solid phase from the unbound material. It is another object of the invention an immunizing and/or vaccine composition comprising at least one of described peptides and a pharmaceutically acceptable carrier and adjuvant. Preferably the adjuvant is a cell, as a dendritic cell. It is another object of the invention a pharmaceutical composition comprising at least one of described peptides and a pharmaceutically acceptable carrier and/or diluent. FIGURE LEGENDS

Figure 1. H A-DR restriction of CD4+ T cells specific for immunodominant sequence p5. CD4+ T cells from the six donors (#1-6) were challenged in 2-day microproliferation assays with p5, in the presence of LCL expressing each of the HLADRBl alleles of the donor. The blanks (i.e.: the basal level of proliferation of CD4+ T cells in the presence of the LCL) are expressed as B+LCL. The data are means of triplicate determination ± SD and are representative of several experiments. Panel A: Donor #1 (HLA-DR8, -DR13); Panel B: Donor #2 (HLA-DR3, -DR7); Panel C: Donor #3 (HLA-DR7, -DR14); Panel D: Donor # 4 (HLA-DR*1101, - DR*1104); Panel E: Donor/Patient #5 (HLA-DR*0405, -DR14), Panel F: Donor/Patient #6 (HLADR* 0403, -DR* 1104). Responses significantly higher than the blanks are indicated as: *p<0,05, **0,00Kp<0,05, ***p<0,001 (determined by unpaired, one-tailed Student's t test).

Figure 2. P5 specific CD4+ T cells recognize the native CEA protein. CD4+ T cells from five donors (#1-5) were challenged in vitro with the CEA protein (20 μg/ml) or human IgG (20 μg/ml) in the presence of autologous APC and tested in 2- day microproliferation assays (Fig. 2A) and/or JNF-a release (Fig. 2B). The blanks (i.e. : the basal level of proliferation or LNF-a release of CD4+ T cells in the presence of autologous PBMC as APC) are expressed as B+APC. The data are means of triplicate determination ± SD and are representative of at least 2 experiments for each CD4+ T cell line. Responses significantly higher than the blanks are indicated as: **0,001<p<0,05, ***p<0,001 (determined by unpaired, one-tailed Student's t test).

Figure 3. Recognition of colon carcinoma cells by p5 specific CD4+ cytotoxic T cells. Panel A. Cytolytic activity, measured in a 51Cr release assay, of HLA-DR13 restricted p5 specific CD4+ T cells from donor #1, against HLA-DR matched or unmatched carcinoma cells expressing CEA. Targets used and their HLA-DR types are indicated in the Figure. The results are representative of several experiments.

Panel B. Western Blot analysis of the expression of CEA by tumor cell lines (Lovo and Kato III) used as targets. Panel C. Cytofluorimetric analysis for surface MHC class II expression in tumor cells used as targets. Filled histograms, stained sample; open histograms, background staining obtained with FIT C-conjugated second-step reagent only.

Figure 4. Cross-reactivity experiments of p5 specific CD4+ T cells for CD66 analog sequences. CD4+ T cells from donor #1 (panel A) and donor/patients #5 (panel B) were challenged with autologous PBMC as APC, pulsed with p5 or synthetic peptides corresponding to the analogs belonging to different members of the CD66 family. The data are representative of at least two experiments for each donor. Responses significantly higher than the blanks are indicated as: ***p<0,001 (determined by unpaired, one-tailed Student's t test). Panel C. Competition experiments: APC from donor #1 were pulsed for 2h with increasing amounts of competitor peptides (1, 5, 10 and 50 μg/ml) and CD4+ T cells were then added in the presence of sub-optimal concentration of p5. Panel E. Alignment of the CD66 analog sequences.

Figure 5. Recognition of peptides corresponding to the subdominant sequences. Figure 6. Recognition of the native CEA protein. Figure 7. Recognition of colon cancer cells expressing CEA by p3-specific CD4+ T cells.

MATERIALS AND METHODS T-cell epitope prediction and peptides' synthesis. The authors selected 11 sequences of the CEA protein for peptide synthesis, based on the TEPITOPE algorithm (23). The authors set the prediction threshold (i.e. the percentage of best scoring natural peptides) at 5%, and selected the sequences predicted to bind at least 40% of the HLA-DR molecules included in the software. The selected sequences were: pl-CEA13-25 (TPWQRLLLTASLL) (SEQ ID. No.l), p2-CEA51-63 (VLLLVHNLPQHLF) (SEQ ID. No.2), p3-CEA99-l 11 (IIYPNASLLIQN) (SEQ ID. No.3), p4-CEAl 17-129 (TGFYTLHVIKSDL) (SEQ ID. No.4), p5-CEA177-189/CEA355-367 (LWWVNNQSLPVSP) (SEQ ID. No.5), p6-CEA425-437 (TYYRPGVNLSLSC) (SEQ ID. No.6), p7-CEA447-459 (YSWLIDGNIQQHT) (SEQ ID. No.7), p8-CEA533-545 (LWWVNGQSLPVSP) (SEQ ID. No.8), p9-CEA568-582 (AYVCGIQNSVSANRS) (SEQ JD. No.9), pl0-CEA652-666 (TYACFVSNLATGRNN) (SEQ ID. No.10) and pi 1-CEA 666-678 (NSIVKSITVSASG) (SEQ ID. No.11).

The sequences were synthesized by manual parallel synthesis as described in (24).

Sequence corresponding to analog sequence CD66al77-189 (LWWINNQSLPVSP) (SEQ ID No. 12) was also synthesized; analog sequence CD66b/cl77-189 was equal to p8-CEA533-545. The peptide purity was verified by reverse-phase high performance liquid chromatography and electron spray mass spectrometry. The synthetic peptides were lyophilized, reconstituted in DMSO at 10 mg/ml, and diluted in RPMI 1640 (GIBCO, Grand Island, NY) as needed.

Subjects and cells. PBMC were obtained from four healthy subjects (donors #1, #2,

#3, and #4) and two colon carcinoma patients (donor/patients #5 and #6). The

Institutional Ethics Committee had approved the study protocol and informed consent was obtained from all healthy subjects and patients before blood sampling. The colon carcinoma Lovo and the gastric carcinoma Kato III cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). LCL used were: Com and Bor, established in our laboratory; PMH-161, BM21, LB, TEM and OLL, kindly provided by K. Fleischhauer (HRS, Milan, Italy); Leis-NTH, the generous gift of F. Marincola (Bethesda, MD); and Pitout, purchased from the European Collection of Cell Culture (Salisbury, UK). The HLA-DR types of donors and tumor cell lines were identified by molecular or serologic typing, and are reported in Table 1. Table 1. HLA-DRB1 type of donors and cells used in this study

Cell type HLA-DRB1

Donors

# 1 PBMC DR*08, DR*13

# 2 ^' PBMC DR*03, DR*07

# 3 PBMC DR*07, DR*14

# 4 PBMC DR*1101, DR* 1104

# 5 PBMC DR*0405, DR*14

# 6 PBMC DR*0403, DR*1104

Cells

Lovo Colon carcinoma DR13

Kato III Gastric carcinoma DR2, DR8

LB EBV-LCL DR13

Pitout EBV-LCL DR*0701

Com EBV-LCL DR*0301

BM21 EBV-LCL DR*1101

TEM EBV-LCL DR*14

Bor EBV-LCL DR*1104

OLL EBV-LCL DR8

Leis NTH EBV-LCL DR*0403, DR*13

PMH-161 EBV-LCL DR*0405

Western Blot analysis. Two millions colon cancer cells were washed twice with Tris-buffered saline (TBS) and lysed with Nonidet P40 (final concentration 1 %) for 30 minute at 4°C in the presence of protease inhibitors. The sample was electrophoresed in 7% polyacrilamide gel and then transferred to nitrocellulose paper. Immunoblotting was performed as described by Towbin, H et al. (25). The blot was incubated with 5% non-fat dry milk in TBS buffer, then with 1 : 1000 dilution of anti-CEA IgG antibody conjugated to peroxidase (the generous gift of Dr.

Rosa, Sorin, Saluggia, Italy) for 1 hour and processed for ECL according to the supplier's instructions. Propagation of CD4+ T cells. Synthetic peptides corresponding to the CEA sequences were pooled and used to stimulate the PBMC from the different donors. Briefly, 20x106 PBMC were cultured for 7 days in RPMI 1640 (GIBCO, Grand Island, NY), supplemented with heat inactivated human serum (10%) (Technogenetics, Milan, Italy), 1-glutamine (2mM), penicillin (100 U/ml), streptomycin (50 μg/ml) (Biowhittaker, Walkersville, MD) (TCM) containing the CEA Pool (1 μg/ml of each peptide). The reactive lymphoblasts were isolated on a Percoll gradient (26), expanded in IL-2 (10 U/ml) containing medium (TCGF, Lymphocult, Biotest Diagnostic Inc., Dreieich, Germany) and restimulated at weekly intervals with the same amount of peptides plus irradiated (4000 rad) autologous

PBMC as APC. CD4+ T cell, clones were obtained by limiting dilution from polyclonal line from donor #2 and #4 and donor/patient #5, as described in (27). Flow cytometry. Cytofluorimetric analyses were performed on a FACStarPlus (Beckton Dickinson, Sunnyvale, CA). The following mAbs were used: anti-CD4-PE and anti-CD8-FITC (Beckton Dickinson), anti-DR (D1.12 hybridoma, ATCC).

FITC-rabbit anti-mouse Ig Ab (DAKO A/S, Glostrup, Denmark) was used as second step reagent in indirect immunofluorescence stainings.

Proliferation assay. CD4+ T cells and autologous irradiated PBMC or HLA-DR matched LCL as APC were diluted at a 1:10 or 1:5 ratio respectively, and used as described in (28). Stimulants were CEA Pool (0.5, 1 and 5 μg/ml), each peptide (10 μg/ml), purified CEA proteins (20 μg/ml) (BiosPacific, Emeryville, CA, Calbiochem, Damstad, Germany) or normal human Ig (20 μg/ml) (Venimmun N, Aventis Behring). Triplicate wells with CD4+ T cells alone and APC alone were used as controls. Three wells with CD4+ T cells plus APC did not receive any stimulus to determine the basal growth rate. In inhibition experiments, mAb D 1.12 or an isotype matched irrelevant mAb (W6/32) (ATCC) was added at 25-50 μg/ml. After 48h the cultures were pulsed for 16h with [3H]TdR (1 mCi, well, 6.7 Ci/mol, Amersham Corp., Milan, Italy). The cells were collected with a FilterMateTM Universal Harvester (Packard) in specific plates (Unifilter GF/C, Packard) and the thymidine incorporated was measured in a liquid scintillation counter (TopCount

NXTTM, Packard). In competition assays, increasing amounts of competitor peptides (LWWINNQSLPVSP or LWWVNGQSLPVSP) (1- 5-10 and 50 μg/ml) were pre-incubated with PBMC as APC for 2h, and then CD4+ T cells were added in the presence of a sub optimal concentration of p5 (5 μg/ml). Cytotoxicity assay. CD4+ T cells were tested for specific lytic activity in a standard 4-h 51Cr release assay as described in (28). The following targets were used: Lovo and Kato III cell lines, unpulsed and p5-pulsed LCL (LB). To allow the expression of MHC class II molecules, tumor cells were cultured for 48 h in the presence of TFN-a (1000 U/ml) (R&D System, Minneapolis). CD4+ T cell stimulation assay. Autologous APC pulsed with purified CEA protein (20 μg/ml) or human IgG (20 μg/ml) were tested for their ability to induce the production of IFN-a by peptides specific CD4+ T cells, after 24-48h of incubation, using a standard ELISA (Biosource Europe, SA, Nivelles, Belgium), following the manufacturers' instructions. RESULTS The authors selected 11 CEA sequences, based on prediction by TEPITOPE, and used a pool of the 11 peptides (CEA Pool) to propagate polyclonal T cell lines from 4 healthy donors (#1-4) and 2 colon carcinoma patients (#5-6) (Table 1). Total PBMC were stimulated with CEA Pool for 7 days, activated cells were expanded in the presence of IL-2 and weekly re-stimulated with irradiated peptide-pulsed autologous PBMC as APC. For all donors, after two or three cycles of stimulation, the authors obtained lines that contained only CD4+ T cells (data not shown). Recognition of p5 by long term polyclonal CD4+ T cell lines. We first tested the response of CD4+ T cells from all donors to the pool of CEA peptides, and then we determined the epitope repertoire of the CEA pool specific CD4+ T cells by testing, at different weeks of propagation of the culture, their prohferative response to each single peptide forming the CEA Pool. At the beginning of the culture CD4+ T cells from the different donors had a larger repertoire; however, after a variable number of weeks of propagation (2-4), depending on the donor, all CD4+ T cell lines strongly and uniquely recognized the CEA sequence repeated at positions 177-189 and 355-367 (ρ5) (Table 2), thus identifying p5 as an immundominant sequence. Table 2. Epitope repertoire of CD4⁺ T cells from six donors propagated in vitro wi CEA Pool^λ.

Donor #1 Donor #2 Donor #3 Donor #4 Donor #5 Donor #6

B+APC 0,1±0 8,5±1,4 10±0,2 11±1,5 5,9±0,9 l±O.l pi 0,2±0 5,9±0,4 11,5±1,3 9,4±1,6 6,9±0,6 0,3±0,1 p2 O±O 3,6±0,8 6±0,6 10,9±1,5 6,7±0,1 0,1±0 p3 0,2±0 10±0,8 9,5±2,6 10,3±1 6,3±0,2 1,4±0

_P4 O±O 6,3±1 11,3±0,8 12,4±8,4 6,8±0,8 0,6±0,1 p5 45±3*** 30±4*** 87±7*** 45±4*** 18±1*** 6±1*** p6 l±O 8,2±0 9,4±1,2 10±1,6 6±2 0,9±0,1 p7 0,1±0 5±0,8 7±0,6 lO±l 4,5±0,7 0,1±0 p8 0,7±0 4,7±0,5 15±3 11±7 5±0,2 0,2±0,1 p9 0,1±0 8,6±2 9±0,5 7 ±1,5 5±3 0,2±0,1 plO 0,2±0 7,4±3 ll±l 15±6 4,6±3 0,9±0,1 pn 0,3±0 8±0,3 9,8±0,4 11±1,7 6±2 0,9±0,3

^APolyclonal CD4⁺ T cell lines from the six donors (#1-6), propagated in vitro with the cEA Pool, were tested with each single peptide (10 μg/ml) forming the Pool in 2- day microproliferation assays. The data, representative ofseveral experiments, are expressed as cpm x 10^"3 and are means of triplicate determination ± SD. Responses significantly higher than the blanks (i.e. : the basal level of proliferation of CD4⁺ T cells in the presence of autologous PBMC as APC: B+APC) were determined by unpaired, one-tailed Student's t test and indicated as: ***p<0,001. HL -DR restriction of p5-specifιc CD4+ T cells.

HLA-DR restriction of p5-specific CD4+ T cells was first verified in vitro by inhibition of their proliferation to the peptide in the presence of an anti-HLA-DR antibody in the culture (data not shown). To identify the HLA-DR restricting alleles for the CEA immunodominant sequence, CD4+ T cells from all donors were challenged in microproliferation assays with LCL, expressing each of the two HLA- DRB1 alleles of the donor, pulsed with p5 (Figure 1). P5 was recognized in association with HLA-DR* 13 by donor #1, HLA-DR*03 and HLA-DR*07 by donor #2, HLA-DR*07 and HLA-DR*14 by donor #3, HLA-DR*1101 and HLA-DR*1104 by donor #4, HLA-DR*0405 and HLA-DR* 14 by donor/patient #5 and HLA- DR*1104 by donor/patient #6. The authors also obtained several p5-specific CD4+ T cell clones from donor #2 that were either HLA-DR3 or HLA-DR7 restricted, from donor #4 either HLA-DR* 1101 or HLA-DR* 1104, and from donor/patient #5 either HLA-DR*0405 or HLA-DR14 (data not shown). The authors cannot exclude that HLA-DRB3 and HLA-DRB4 molecules in addition to HLA-DRB1 can also present the CEA immunodominant sequence to the polyclonal CD4+ T cell lines. Recognition of native CEA by p5-specific CD4+ T cells. To verify whether p5 contains naturally processed epitope(s), the authors tested the recognition of the native CEA protein both after processing and presentation by autologous APC after phagocytosis of purified CEA protein, and directly by recognition of carcinoma cells expressing endogenous CEA and MHC class II molecules (Figures 2 and 3). CD4+ T cell lines from five donors (#1-5) were challenged with autologous APC pulsed with the CEA protein and assayed for 3H- thymidine incorporation (Fig. 2A) and/or LNF-a release (Fig. 2B). CD4+ T cells from all tested donors strongly and significantly recognized the CEA protein while they did not recognize normal human IgG, demonstrating their CEA specificity. Since CD4+ T cells produce large amount of TNF-a, the authors tested their lytic activity. As shown in Figure 3, CD4+ T cells that recognized p5 in association with HLA-DR13 (see Fig. 1A), killed DR13-LCL pulsed with p5 and, most importantly, Lovo cells expressing CEA and HLA-DR13; while they did not kill Kato HI cells, expressing CEA and unrelated HLA-DR alleles, or unpulsed HLA- DR13-LCL (Fig.3A). The levels of expression of CEA (Fig. 3B) as well as of surface MHC class II molecules (Fig. 3C), after 48h culture in the presence INF-a, by tumor cells are also shown.

P5- specific CD4+ T cells do not cross-react with analog self-sequences present on homologous proteins of the CD66 family. Since CEA, or CD66e, belongs to a family of highly homologous proteins that are expressed at high levels in normal hematopoietic cells, the authors verified the sequence similarity of p5 among the different CD66 molecules. Two sequences that differ from p5 for only one amino acid (substitutions I V and G N, respectively) were found. The first analog (LWWINNGQSLPVSP) is present in the CD66a molecule at residues 177-189. The second (LWWVNGQSLPVSP) is present in the CD66b and CD66c molecules, at residues 177-189. This latter sequence is also present in the CEA or CD66e molecule at residues 533-545 (p8), and is comprised in the CEA Pool used to propagate the CD4+ T cell lines and never elicited prohferative activity in any donor. Figure 4 shows the results of the experiments of cross-reactivity of p5- specific CD4+ T cells from donor #1 and donor/patient #5 in the presence of the two CD66 analogs. CD4+ T cells proliferated in the presence of p5 but not in the presence of the analogs, demonstrating that recognition of p5 is indeed CEA specific (Fig. 4A and B). To discriminate whether the lack of cross-recognition was due to poor binding to the HLA-DR molecules or to lack of T cell receptor (TCR) stimulation, the authors performed competition experiments in which APC prepulsed with increasing amount of competitor peptide (1, 5, 10 and 50 μg/ml) were used in microproliferation assays to stimulate CD4+ T cells from donor #1 in response to a sub-optimal dose (5 μg/ml) of p5 (Fig. 4C). The response of CD4+ T cells to p5 decreased, in a dose dependent manner, in the presence of peptide CD66al77-189, while it was only marginally affected in the presence of peptide CD66b/cl77-189 (p8). These results demonstrate that peptide CD66al 77-189 competes with p5 for binding to the HLADR molecules but the MHC-peptide complex is not recognized by the TCR of p5-specific CD4+ T cells, while peptide CD66b/cl77-189 (p8) appears to be a poor binder. Repertoire of subdominant CEA epitopes. To study the existence of subdominant CEA epitopes, peripheral blood mononuclear cells from donor #2 and donor #3 were stimulated in vitro with a pool of CEA peptides containing 10 peptides (pi, p2, p3, p4, p6, p7, p8, p9, plO), i.e. in the absence of the immunodominant sequence p5. The repertoire of recognized peptides was tested in microproliferation assays, by testing the recognition of each single peptide by polyclonal CD4+ T cell lines. CD4+ T cells from donor #2 proliferated in the presence of p3, p6 and p9, while CD4+ T cells from donor #3 proliferated in the presence of p3 and pll. Recognition of the peptides corresponding to the subdominant sequences was confirmed in different experiments and it was stable during time (Fig. 5). Generation of peptide-specific CD4+ T cell clones

Polyclonal CD4+ T cell lines from both donors were cloned by limiting dilution and we obtained p3-, p6- and p9-specific CD4+ T cell clones from donor #2, and p3- and pi 1 -specific CD4+ T cell clones from donor #3. Single clones are used to test the recognition of the naturally processed sequences by peptide-specific CD4+ T cells. Recognition of the native CEA protein. p3-specific clone 11 was tested for recognition of the purified CEA protein.

Recognition of the native sequence, by p3-specific CD4+ T cells, was tested by ELISA as gamma-IFN release in the presence of the peptide and two different CEA proteins. CEA biospacific was purified from a colon cancer cell line and rCEA fragment, a recombinant fragment of CEA that does not include the p3 sequence was produced. Clone 11 specifically produced gamma-IFN in the presence of p3 and in the presence of the CEA protein (biospacific), while it did not recognize the negative control (rCEA fragment) (Fig. 6).

Recognition of colon cancer cells expressing CEA by p3-specifϊc CD4+ T cells. Three p3-specific CD4+ T cells clones from donor #3 were tested for recognition of colon carcinoma cells expressing CEA and the HLA-DR14 restricting allele.

Although at different levels, all the three clones specifically produced gamma-IFN in the presence of colon cancer cells (Calu-1), while they did not recognize HLA-DR mismatched colon cancer cells expressing CEA. These experiments further demonstrate that p3 contains a naturally processed epitope(s), expressed at the cells surface of colon cancer cells in association with MHC class II molecules (HLA-

DR14) (Fig. 7). DISCUSSION

In this study the authors used a combined approach of bioinformatics followed by in vitro validation with biological assays to identify the amino acid sequence repeated at residues 177-186 and 355-367 (p5) on the CEA protein as a strongly immunogenic and immunodominant region that contains naturally processed epitope(s). The ability of the immune system to focus on a selected number of epitopes of a complex antigen is a distinctive feature of most T cell immune responses, and it is termed immunodominance. The identification of immunodominant epitopes on tumor antigens is of particular importance for vaccine development to increase the number of patients eligible for therapy. Immunodominance may be dictated by antigen processing mechanisms, which may vary in different cell types, by intermolecular competition for MHC binding, by HLA-DM molecules and by the existence of a biased T cell repertoire (29, 30). In the case of the CEA, the epitope immunodominance may also be related to the high degree of glycosylation of the molecule. Indeed, CEA is a glycoprotein of 180 Kda mw that comprises 60% carbohydrate with twenty-eight potential glycosilation sites. Because of this, available linear sequences potentially able to form CD4 epitopes, are probably significantly reduced compared to non-glycosilated proteins. Indeed, recently the authors identified four immunodominat regions on the tumor specific antigen MAGE-3, which is a non-glycosilated protein of 74 Kda mw (31). It cannot be excluded that other mechanisms besides glycosylation may account for the different characteristics in the T helper response seen between the two tumor antigens. Nonetheless, using different experimental approaches, other immunodominat as well as subdominant linear and glycopeptidic CEA epitopes able to activate CD4+ T cell responses might also be found. The existence of MHC class II restricted immunogenic glycopeptides has been previously documented for the melanoma antigen tyrosinase (32) and the tumor antigen MUCl (33).

Recently, a CEA helper epitope at residues 653-667, able to induce proliferation of CD4+ T cells in association with HLA-DR4, -DR7 and -DR9, was identified (22). This sequence largely overlaps p 10-CEA652-666 that is comprised in the CEA Pool used in our study, and that never elicited CD4+ T cell responses from any subject studied. This discrepancy may be explained by the strong immunodominance of p5, which was not tested in (22), and it suggests that sequence CEA653-667 may contain subdominant epitope(s). The authors showed that p5 contains naturally processed epitope(s), and the native sequence(s) could be produced through both the exogenous and the endogenous processing pathways. In fact, native CEA177-186/355-367 was produced and presented by APC after uptake and processing of the soluble protein and directly by HLA-DR molecules on tumor cells. In this case, the epitope may be produced either in the cytoplasm and reach the endosomal/lysosomal compartment through a leakage from the endogenous pathway or directly in the endosomal/lysosomal compartment from CEA recycling from the plasma membrane.

A fundamental feature of the epitope(s) identified in our study is the strict specificity for CEA. CEA belongs in fact to a family of highly homologous proteins, some of which are expressed in normal cells (i.e. granulocytes, monocytes, dendritic cells, and activated T and B lymphocytes). Therefore, the use of CEA as vaccine has the potential to induce autoimmune responses against normal hematopoietic cells. The generation and availability of immunogenic self-epitopes during ontogeny impinge directly on the induction of central and peripheral tolerance and the consequent generation of a responsive T cell repertoire. The CEA, although not expressed directly into primary or secondary lymphoid organs, share extensive sequence homology with members of the CD66 family expressed within the lymphoid tissue. CD4+ T cells are thus exposed to several CEA analog sequences during ontogenesis. This process may well lead to the induction of tolerance against these epitopes, resulting in a marked shaping of the CEA specific peripheral CD4+ T cell repertoire towards a limited number of CEA epitopes, which are not encountered during ontogenesis. The authors showed that, although the immunodominat CEA epitope differs from the analog self-sequences for only one amino acid, its recognition is very selective. Indeed, one (CD66b/cl77-189) of the two competitor peptides bound the MHC class II molecules very poorly. The other (CD66al77-189) bound the MHC complex at high affinity, however the MHC-peptide complex could not be recognized by the TCR of the p5 specific CD4+ T cells. CD4+ T cells specific for the analogs have been probably deleted either in the thymus or in the periphery. On the contrary, the repertoire of p5 specific CD4+ T cells is present both in normal donors and colon cancer patients and the TCR of these cells does not allow self antigens recognition through molecular mimicry, resulting in a repertoire of CD4+ T cells specific for CEA. Indeed, the lack of cross-reactivity of p5 specific CD4+ T cells for the analogs may be considered an additional mechanism of self-tolerance. A lack of cross-reactivity with homologous self-proteins (Kallikrein) by prostate- specific antigen cytotoxic T cells was previously demonstrated (34), pointing to possible similar mechanisms of immune tolerance in both models. Recently, the induction of an anti-CEA response in the absence of autoimmunity was demonstrated in CEA transgenic mice vaccinated with recombinant vaccinia virusexpressing CEA (35). Nonetheless, it will be important to address the question of the possible induction of autoimmunity in clinical trial settings. In conclusion, the strong immunodominance and the lack of cross-reactivity in vitro of CD4+ T cells for analog self-sequences, potentially expressed on hematopoietic cells, make the identified immunodominat CEA sequence an excellent candidate for peptide based immunotherapeutic intervention in patients bearing CEA positive tumors. Moreover, it represents a valuable tool to characterize the natural anti-CEA CD4+ T cell response as well as to monitor the anti-CEA CD4+ T cell response before and after vaccination employing different strategies (18, 36-38).

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Claims

1. An isolated CEA class II binding peptide able to react with CD4+ T cells having an amino acid sequence comprised in the sequences of peptides of the following list: SEQ ID. No.l, SEQ LD. No.2, SEQ ID. No.3, SEQ ID. No.4, SEQ ID. No.5, SEQ JD. No.6, SEQ JD. No.7, SEQ ID. No.8, SEQ ID. No.9, SEQ ID. No.10 and SEQ JD.

No.ll.

2. An isolated CEA class II binding peptide able to react with CD4+ T cells according to claim 1 having an amino acid sequence comprised in the sequences of peptides of the following list: SEQ JD. No.2, SEQ ID. No.3, SEQ ID. No.5, SEQ JD. No.7 and SEQ ID. No.ll.

3. An isolated CEA class II binding peptide able to react with CD4+ T cells according to claim 2 having an amino acid sequence comprised in the sequences of peptides SEQ JD. No.3 or SEQ ID. No.5.

4. An isolated nucleic acid encoding the peptide according to any of previous claims. 5. An expression vector able to efficiently express the isolated nucleic acid of claim

4.

6. A host cell transformed with the expression vector of claim 5.

7. A multimer class π MHC complex comprising at least one peptide according to claim 1. 8. The multimer class II MHC complex comprising at least one peptide according to claim 1 wherein the multimer is a tetramer.

9. An. antigen presenting cell expressing at least one of peptides according to claim 1.

10. A method for detecting CD4+ T cells reacting with at least one of peptides of claim 1 in a biological sample comprising the steps of: - exposing the biological sample to the multimer complex of claim 7 or 8 in a condition allowing the binding of the multimer complex to cells; - detecting reacted cells by means of a revealing system.

11. The method according to claim 10 wherein the revealing system consists of a detectable molecule bound to the multimer complex of claim 7 or 8. 12. The method according to claim 11 wherein said detectable molecule is a chromophore.

13. The method of claim 10 wherein the revealing system consists in the detection of molecules produced by CD4+ T cells upon activation by the peptide of claim 1, by reacting with specific molecule reagents, as antibodies and revealing means.

14. The method of claim 13 wherein the molecules produced by CD4+ T cells upon activation by the peptide of claim 1 belong to the following group: performs, granzyme and cytokines.

15. The method of claim 14 wherein cytokines are gamma-IFN, IX- 10, IL-4, LL-5.

16. A method for isolating CD4+ T cells reacting with at least one of peptides of claim 1 in a biological sample comprising the steps of: - exposing the biological sample to the multimer complex of claim 7 or 8 bound to a chromophore in a condition allowing the binding of the multimer complex to cells;

- separating reacted cells by FACS.

17. A method for isolating CD4+ T cells reacting with at least one of peptides of claim 1 in a biological sample comprising the steps of: - exposing the biological sample to the multimer complex of claim 7 or 8 in a condition allowing the binding of the multimer complex to cells;

- exposing the reacted sample to a solid phase-reagent specific for molecules produced by CD4+ T cells upon activation by the peptide of claim 1 ;

- separating the solid phase from the unbound material. 18. An immunizing and or vaccine composition comprising at least one of peptides according to claim 1 and a pharmaceutically acceptable carrier and adjuvant.

19. The composition according to claim 18 wherein the adjuvant is a cell, as a dendritic cell.

20. A pharmaceutical composition comprising at least one of peptides according to claim 1 and a pharmaceutically acceptable carrier and/or diluent.