human t-cell responses to hla-a-restricted high binding ...human t-cell responses to...

788s Vol. 7, 788s-795s, March 2001 (Suppl.) Clinical Cancer Research

Human T-Cell Responses to HLA-A-restricted High Binding Affinity Peptides of Human Papillomavirus Type 18 Proteins E6 and E71

M i c h a e l P. Rudol f , S t ephen M a n ,

Corne l i s J. M. Mel ie f , A le s sandro Sette, and

W. M a r t i n Kast 2

Cardinal Bernardin Cancer Center, Loyola University of Chicago, Maywood, Illinois 60153 [M. P. R., W. M. K.]; Department of Medicine, University of Wales College of Medicine, Cardiff CF14 4XX, Wales, United Kingdom [S. M.]; Department of Immunohematology and Blood Bank, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands [C. J. M. M.]; and Epimmune, Inc., San Diego, California 92121 [A. S.]

Abstract Human Papillomaviruses (HPVs) are sexually trans-

mitted pathogens, which are implicated in the etiology of cervical cancer. The early proteins E6 and E7 of HPV have transforming capacity and interfere with the cell cycle control of infected host cells and are essential for the maintenance of the transformed state. Identification of MHC class I-restricted, immunogenic peptides derived from either the E6 or the E7 protein is essential for the design of vaccines as well as the monitoring of clinical trials and immunotherapeutic approaches for the treatment of HPV-18-induced carcinomas. We have determined the binding affinities for all possible 9-mer peptides spanning the entire E6 and E7 amino acid sequence for the HLA-A*0101, HLA-A*0201, HLA-A*0302, HLA- A*l102, and HLA-A*2402101 molecules by a competition assay with reference peptides, thereby establishing the binding peptides as potential cytotoxic T-cell epitopes. From the HLA-A*0201 binding peptides, we selected five E6-derived and one E7-derived peptide with high affinities for HLA-A*0201. These six peptides were tested for their immunogenicity by in vitro immunization assays with purified human CD8+ T cells. We identified three HPV-18 E6-derived peptides (ELTEVFEFA, KTVLELTEV, and KLPDLCTEL) and the E7-derived peptide TLQDIVLHL to be highly immunogenic. Overall, these results will help to design vaccines for the prevention or treatment of HPV-18-induced cervical cancer.

This work was partially supported by a fellowship from the Illinois department of Public Health, Office of Womens Health, and NIH Grants RO1 CA74397 and PO1 CA74182. S. M. is a Royal Society University Research Fellow. z To whom requests for reprints should be addressed, at Cardinal Ber- nardin Cancer Center, Loyola University of Chicago, 2160 South First Avenue, Maywood, IL 60153. Phone: (708) 327-3325; Fax: (708) 327- 3238, E-mail: [email protected].

Introduction HPVs 3 are very common sexually transmitted viruses. In

some populations, it is suggested that the majority of sexually active women are exposed to high-risk HPV types (1, 2), and

1% of all cervical intraepithelial neoplasia (precancerous lesions) progress to squamous cell carcinoma. HPVs are involved in the etiology of cervical carcinoma, with up to 10% of total mortality attributable to cancer worldwide (3). More than 70 different genotypes of HPVs have been described, of which the HPV types involved in the etiology of cervical cancer are predominantly HPV types 16 and 18, and less frequently HPV types 31, 33, 35 and 45 ( l , 4). These HPV types are considered to be high-risk viruses for the development of cervical carcinoma, and viral DNA of these types can be detected in >99.8% of all cervical carcinomas (5). Worldwide, 50% of the tumors are accounted for by HPV type 16 and a lower portion of 14% is accounted for by HPV type 18. However, HPV type 18 predominates in Asian women as well as in certain types of cancer-like adenocarcinomas and adenosquamous carcinomas (1). Moreover, cervical carcinomas induced by HPV-18 are more aggressively growing, have a poorer prognosis, and the overall survival rate is significantly lower than for HPV-16 (6, 7).

The mechanism by which the transformation of the infected cells occurs was studied extensively in the past (8). The two viral proteins E6 and E7 bind to the tumor suppressor gene products p53, pRb, and Rb-like proteins, respectively, thereby interfering with the regulatory function of the cell cycle control proteins. This contributes to transformation of the infected cells. Both proteins, E6 as well as E7, are retained and highly expressed in vivo in all stages of cervical carcinoma and, therefore, can be considered as tumor-specific antigens for HPV-induced cervical cancer.

CTLs have been implicated in control of tumor growth and virus-infected cell rejection in HPV-infected patients (9-11). Moreover, it has been shown in animal models that vaccination with MHC class I-restricted, specific CTL epitopes derived from tumor antigens induces a highly effective CTL response capable of preventing tumor growth or eradicating existing tumors in

vivo (12-14). Therefore, the identification of tumor antigen- derived immunogenic peptides binding with high affinity to human MHC class I molecules allows the design of potential vaccines as well as accurate monitoring of immune responses in patients in vaccine trials (15-20).

Computer programs have been designed to predict potential CTL epitopes based on the primary amino acid sequence of

3 The abbreviations used are: HPV, human papillomavirus; PBL, peripheral blood lymphocyte; IL, interleukin; GM-CSF, granulocyte/ macrophage-colony stimulating factor; DC, dendritic cell; FACS, fluo- rescence-activated cell sorter; HBc, hepatitis B core.

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Clinical Cancer Research 789s

the tumor antigen and MHC haplotype binding motifs (21-23). Nevertheless, the prediction of peptides based on MHC class I binding motifs by computer programs alone does not necessarily correlate with the binding affinity of the peptides to the MHC class I molecule. Therefore, it is important to perform binding assays to identify candidate epitopes with high binding affinity to MHC class I molecules, which otherwise might be missed by the prediction algorithms.

The ability of a peptide to induce a CTL response is determined by several factors, ranging from binding affinity, amino acid composition, complex stability, and the T-cell rep- ertoire. Therefore, the immunogenicity of the potential CTL epitopes has to be tested in immunization assays in vitro on human PBLs. Such in vitro immunization assays have been widely used before to identify potentially immunogenic CTL epitopes of various tumor antigens (24-26).

For cervical cancer, the most prevalent type of papillomavirus detected is HPV-16, and therefore, most of the efforts to identify CTL epitopes thus far have concentrated on this virus type. Only recently two papers have been published identifying potential CTL epitopes derived from HPV-18 E6 proteins, either by eluting a peptide from a tumor cell line (27) or from in vitro immunization assays with human PBLs and synthetic, computer-predicted potential CTL epitopes specific for HLA-A*0201 molecules (28). Here we report the binding affinities for 9-mer peptides derived from HPV-18 E6 and E7 proteins for the HLA-A*0101, HLA-A*0201, HLA-A*0302, H L A - A * l l 0 2 and HLA-A*2402101 molecules as determined by screening a set of all possible 9-met peptides spanning the entire amino acid sequence of the two proteins in a competition assay for binding with a labeled reference peptide to purified human MHC class I molecules. Subsequently, we tested six peptides binding to HLA- A*0201 with high affinity for immunogenicity in vitro with purified CD8+ T cells from normal donors. Our test assay allowed us to calculate CTL precursor frequencies in normal human donors for each of the peptides tested, and for five of the six peptides, we found frequencies comparable with a hepatitis B-derived peptide known to be immunogenic in

vitro as well as in vivo. In conclusion, we were able to identify three peptides derived from E6 and one peptide derived from E7 to be immunogenic and to obtain a reasonable pCTL in healthy donors, rendering these peptides suitable candidates for the development of a peptide vaccine for HLA-A*0201-positive humans.

Materials and Methods Peptides. A total of 150 peptides derived from HPV-18

E6 and 97 peptides from E7 were synthesized by Fmoc chemistry by solid-phase strategies on a multiple peptide synthesizer AMS 422 (Abimed, Langenfeld, Germany). Pep- tides were >90% pure by reverse-phase high-performance liquid chromatography. Peptides were lyophilized and kept at room temperature until use. Peptides were diluted either in PBS/3% DMSO (Sigma Chemical Co., St. Louis, MO) at 1 mg/ml and kept at - 80~ or peptides were dissolved in 100% DMSO at 50 mg/ml and stocks were kept at - 80~

subsequently, stock solutions were diluted into PBS at pH 7 to 10 I~g/ml final concentration.

Reference peptides were YLEPAIAKY for HLA-A*0101, FLPSDYFPSV for HLA-A*0201, KVFPYALINK for HLA- A*0302, AVDLHFLK for HLA-A*ll02, and AYIDNYNKF for HLA-A*2402101. Peptides were 1251 radiolabeled by the chloramine T method (29).

Peptide-MHC Binding Assay. Peptide binding assays were performed as described earlier (30). Briefly, peptide-MHC binding assays were designed as a competition assay for binding to recombinant, purified MHC class I molecules between the peptide to be tested and a radiolabeled reference peptide. The HLA-A*0101, HLA-A*0201, HLA-A*0302, HLA-A*l l02, and HLA-A*2402101 molecules were isolated from EBV-transformed cell lines Steinlin, JY, GM3107, BVR, and KT3, respectively. Cells were lysed by NP40, purified by chromatography over Sepharose columns, and subsequently further purified by affinity chromatography with antibodies B1.32.2 (anti-HLA-B and anti-HLA-C) and W6/32 (anti-MHC class Iot chain). The amount of MHC molecules resulting in binding of 15% or more radiolabeled peptide (normally in the range of 10-50 nM) was incubated in 0.05% NP40/PBS with 5 nM radiolabeled peptides and titrated amounts of unlabeled competitor peptide (usually in the range of 10 lxg to 1 ng/ml) in the presence of 1 IxM human [32-microglobulin (Scripps Laboratories, San Diego, CA) and protease inhibitors. After 48 h at 23~ bound radioactivity was determined by size exclusion chromatography on TSK 2000 gel filtration as described (31). Peptides were tested at one to two high doses, and the 50% inhibitory dose (ICso) of peptides yielding positive inhibition was determined in subsequent experiments. To allow comparison of results from different experiments a relative binding figure (rBI) was calculated for each peptide by dividing the ICso of the positive control for inhibition of unlabeled probe peptides by the ICso for each peptide tested. The ICsos for the standards were 81 nM for A*0101, 5 nM for A*0201, 30 nM for A*0302, 9 nM for A l l and 22 nM for A'2402101.

Donor Material. PBLs were obtained from HLA-*0201 positive healthy donors by apheresis. Cells were isolated by Lymphoprep gradient (Nycomed Pharma, Oslo, Norway), ali- quoted, and kept in liquid nitrogen until use. Donors were not tested serologically for the presence of antibodies against HPV proteins. Nevertheless, because of the very low prevalence of HPV-18 in the United States (with an overall risk of contracting genital papillomaviruses of 2 -8% for men of 10-20% for women; with < 10% of all cervical cancer specimens containing HPV-18 DNA; Refs. 1 and 4), we expect less than one of the four donors to be positive for HPV-18.

In Vitro Immunization Assay. Dendritic cells were gen- erated from frozen PBLs as described earlier (32). Briefly, cells were thawed and incubated for adherence in tissue culture flask in RPMI 1640 (0.1 mM MEM nonessential amino acids, 1 mM pyruvic acid, L-glutamine, and 50 txg/ml kanamycin) containing 5% human AB serum (Sigma) for 2 h at 37~ Nonadherent cells were carefully washed away, and adherent cells were incubated at 37~ in RPMI + 5% AB serum containing 1000 units/ml IL-4 (Peprotech, Rocky Hill, N J) and 800 units/ml GM-CSF (Immunex, Seattle, WA). After 7 days, cells were harvested and incubated for 72 h in RPMI + 5% AB serum



790s Potential HPV-18 E6/E7 CTL Epitopes

Table 1 Binding properties of HPV-18-derived peptides to MHC class I molecules

Position ICso Position ICso in E6 nM in E7 nM

HLA-A*O101 HLA-A*0101

8i =90' [Y'S L T

l 95 ~ 104 tD I

HLA-A*0201

47-56 IFI~ FKtDLF]V

DiS]ViYIGID~ t88

E I T C V 506 ~[LlYIglLILm 5O6 DIL1FIV[VI~f~ 736

810 900 988

V LiE LiT E ~ 106 iYN=i~L i R,C 135 I D F Y I S R i 417

40-48 l E V I E V F E F ] 500 Di L F V V YI R 500

24- 37 lsi~ Q D i iE ~ TiC 532 13-21 JKIL_PDLiC:T~g 602

[ ~ ~s;<~JDt7 E I "r Icv Y ~ 649 [:~: ~';~,]L[Y]NL L[i RC]~ 676

HLA-A*0302

H L N E K R [ 188 T c v Y c _ ~ 2oo F YS R I R 273 , N EL IR C 3~3 L F V V u ~ 330 VY!RDS I 441 I T ICVu 462 : R L[Q R R R~ 484 R C~LLRC Q 508 C L i R C Q K 526 I E!I 3"[C ~ 625 E KIRRiF H 667 L I n C h . R 833 VVWRIE S 1000

HLA-A*ll02

G D ~ E ~ 2

F u 27 H S C C N R 64 N L L I Re 188 __. E F-A ~ 257 C L R C ~ K 375 L F VV YR 500

L I R C L R 529 K P LN P A 563

g"l VL EL 957

HLA-A*2402101

100

185-94 IVI~OIDITILIFIr~ 222

HLA-A*0201

7 - t 5 T T [ Q ! D I V L ~ I 86-94 F~QLF[LNTIF 417 89-97 L F L N T [ L S F ] ' ~ 820 90-98 F ~ N T L [ S F V C 943

HLA-A*0302

[ S9:g8 l~qLlcNclcV/t 73

HLA-A* 1102 ~ 5 I 6 15 IAI~ILIQtDIIIvlLN 12

HLA-A*2402101

] 58- 67 R ] H T I ~ L c I ~ c c 733

Framed amino acids indicate optimal anchors; amino acids in italic indicate tolerated amino acids at anchor position. Shading indicates poor solubility of the peptide in PBS.

containing 25% monocyte-derived conditioned medium + 800 units/ml GM-CSF. Monocyte-derived conditioned medium was prepared by incubating adherent cells with 0.001% Pansorbin cells (Calbiochem, La Jolla, CA) for 24 h. Supernatant was centrifuged, filtrated, and kept at -20~

CD8+ T cells were isolated from nonadherent PBLs by incubating cells with anti-CD8 Dynabeads (Dynal, Oslo, Nor- way) in PBS + 1% AB serum for 1 h on ice and subsequently selected by magnetic force. After six washes, beads were incubated with Detachabead reagent (Dynal) for 1 h at room temperature, and empty beads were separated from cells by magnetic selection. Cells were washed in RPMI + 5% AB serum and used for in vitro immunization assay.

DCs were loaded with 40 ~xg/ml peptide and 3 Ixg/ml [32-microglobulin (Biodesign, Kennebunk, ME) for 4 h on ice, washed, irradiated with 25 Gy, and mixed with autologous CD8+ T cells at ratio 1:10. Cells were distributed into 48-well plates (Costar, Cambridge, MA) in 500 ~xl/well RPMI + 5% AB serum and 10 ng/ml IL-7 (Peprotech). After 7 days, medium was removed, and cells were transferred into a new plate containing irradiated peptide-loaded adherent autologous cells. Cells of each well were transferred individually in 500 Ixl/well RPMI + 5% AB serum. After 24 h, l0 ng/ml IL-10 (Peprotech) was added, and after 48 and 96 h, 20 units/ml IL-2 (Peprotech) were added. Restimulation was repeated after 14 days as described above.

Cytotoxicity Assay. HLA-A*0201-positive JY cells were labeled with 51Cr (DuPont NEN, Boston, MA), washed, and loaded with peptides. Effector cells were mixed with 2 • 103 peptide-loaded or unloaded JY cells and 20 • 103 K562 cells (natural killer blocker cell line) in 96-well plates (Costar). Plates were incubated for 6 h at 37~ and 50 txl/well supernatant was transferred into OptiPlate (Packard Instrument Co., Meriden, CT), mixed with Microsynth-40 (Packard), and counted in Counter (Packard) for 1 min/well. Spontaneous and maximal release were determined by incubation of labeled JY cells in medium or 1% Triton X-100, respectively. Lysis was calculated according the following formula: % lysis = [cpm (sample) - cpm (spontaneous release)]/[cpm (maximal release) - cpm (spontaneous release)], and specific lysis was calculated as: % specific lysis = % lysis (peptide loaded targets) - % lysis (unloaded targets). A well was counted as "positive" when the percentage of specific lysis was >10%.

Calculation of Precursor Frequency. The precursor frequency pCTL was calculated by dividing the total number of CD8 + T cells/plate by the number of positive wells, assuming that a positive CTL response in a well of the 48-well plate was derived from a single responding cell in the primary stimulation (the probability of 90% of a monoclonal response in a total amount of 24 • 106 CD8 + T cells in the primary induction would require no more than 0.2-0.3 specific precursor cells/ well; Ref. 33).

Results Binding Affinities for 9-mer Peptides to HLA-A Mole-

cules. A set of 9-mer peptides overlapping by 8 amino acids and spanning the entire amino acid sequences of HPV-18 E6 and E7 proteins were synthesized and analyzed for binding to




Table 2 Immunogenicity and precursor frequencies of HLA- A*0201-restricted peptides as determined by in vitro immunization

assays with human CD8 + cells purified from normal donors

% specific lysis % positive Precursor Peptide Donor (average) " wells b frequency C

LQDIEITCV 1 (female) 21.5 6.3 7.7 X 10 6

(E6) 2 (female) 17.3 8.3 ICso 86 nM 3 (male) 12.7 2

4 (male) 37.0 10.4 ELTEVFEFA 1 (female) 17.8 43.8 1.6 • 10 6

(E6) 2 (female) 14.5 29.2 ICso 500 nM 3 (male) 21.3 31.3

4 (male) 15.8 20.8 KTVLELTEV 1 (female) 15.5 10.4 3.6 • 106 (E6) 2 (female) 16.6 4.2 ICso 106 nM 3 (male) 18.5 35.4

4 (male) 15.7 8.3 KLPDLCTEL 1 (female) 18.2 27.1 3.2 • 106 (E6) 2 (female) 35.2 4.2 ICso 602 nM 3 (male) 28.9 20.3

4 (male) 15.5 12.5 FAFKDLFVV 1 (female) 29.0 10.4 6.3 • 106 (E6) 2 (female) 0.0 0.0 IC5o 3 nM 3 (male) 13.2 12.5

4 (male) 13.9 10.4 TLQDIVLHL 1 (female) 13.9 10.4 3.1 • 106 (E7) 2 (female) 16.4 20.8 iC5o 1 nM 3 (male) 20.8 41.7

4 (male) 13.5 10.4

a Average percentage of lysis of wells showing >10% specific lysis.

b Number of wells with specific lysis > 10% divided by 48 (number of wells/plate).

~ Precursor frequency indicates the number of purified CD8 + T cells containing one specific CTL. Precursor frequency was calculated by the assumption that responses in positive wells/48-well plate were derived from a single responding cell in the primary stimulation. The number is determined by the total amount of CD8 + T cells used in the four experiments per peptide divided by the total number of positive wells from all four experiments. The HBc peptide has a precursor frequency of one specific CTL in 1.6 • 106 purified CD8 + with an AVG of 25.2% specific lysis as determined by using the same method above on the same donors.

recombinant MHC class I molecules of HLA-A*0101, HLA- A*0201, HLA-A*0302, H L A - A * l l 0 2 , and HLA-A*2402101 haplotypes. Each peptide was tested for binding to each recombinant haplotype in competition with a radiolabeled reference peptide for the corresponding haplotype. The results are summarized in Table 1. It has been found that an ICso threshold of - 5 0 0 nM determines the capacity of a peptide epitope to elicit a CTL response in vitro (34). To not miss a potential CTL epitope, we set the threshold at 1000 riM, and we show in Table 1 only peptides with an ICso of 1000 ng or less. This method allowed us to identify a total of 8 HLA-A*0101 binding peptides (8 of E6 and none of E7), 16 HLA-A*0201 binding peptides (12 of E6 and 4 of E7), 16 HLA-A*0302 binding peptides (15 of E6 and 1 of E7), 14 H L A - A * l l 0 2 binding peptides (12 of E6 and 2 of E7), and 4 HLA-A*2402101 binding peptides (3 of E6 and 1 of E7) with an ICso of 1000 nM or less. Peptides not listed had an ICso of < 1000 nM or did not bind at all to any of the HLA-A haplotypes.

We also included available information about anchor residues into Table 1 (30). Amino acids matching the required

anchor residues as well as tolerated anchor residues of each peptide were highlighted. It has to be noted that not all peptides with a low ICso have a binding motif present (determined by either two preferred amino acids as anchor residues or one preferred and one tolerated amino acid). Most of the peptides without a clear binding motif were not readily soluble and a nonspecific inhibition of the reference peptide attributable to this fact may have contributed to the low ICso (shading indicates peptides, which were not easily soluble in water and had to be dissolved by adding increasing amounts of DMSO).

Overall, the binding studies allowed us to identify high- affinity binding peptides and to limit dramatically the number of peptides subsequently to be tested in the laborious and more t ime-consuming in vitro immunization experiments as compared with the suggested number of peptides to be tested by computer program predictions (e.g., for HLA-A*0201 we would have had to test 40 peptides to cover all high-affinity binding peptides).

In Vitro Immunization Assay with Human PBLs. For subsequent experiments, we focused on HLA-*0201-restricted peptides with an IC5o of 600 nM or lower (see Table 1), which contained the required anchor residues and had a good solubility in PBS. The E6-derived peptides GLYNLLRIC and KCIDFYSRI were not included because of solubility problems, and peptides SLQDIEITC and FKDLFVVYR of E6 as well as FQQLFLNTL of E7 were not included because of missing anchor residues. The remaining six peptides LQDIEITCV, ELTEVFEFA, FAFKDLFVV, KTVLELTEV, and KLPDLC- TEL of E6 and TLQDIVLHL of E7 were tested in an in vitro

immunization assays with four healthy, HLA-A*0201-posit ive donors (2 male, 2 female). From each donor, DCs were gener- ated by incubation of adherent mononuclear cells for 7 days in IL-4 and GM-CSF, followed by a maturation step with a monocyte-derived conditioned medium. Resulting activated DCs (pu- rity of > 9 0 % as determined by staining for CD83 in FACS analysis; data not shown) were loaded with peptides, irradiated, and mixed with autologous, purified CD8 + T cells at a ratio of 1:10. After two rounds of restimulation after 7 days and 14 days (cells of each well were restimulated independently from all of the other wells), the remaining cells of each well were collected and tested independently in a cytotoxicity assay by splitting in three portions: one-third of the cells were used as effector cells on unloaded target cells; one-third of the cells were used as effector against peptide-loaded target cells; and the remaining one-third of cells was used for further restimulation. Results from the in vitro immunization assays and cytotoxicity assays are summarized in Table 2. The average percentage of lysis was calculated by averaging the sum of the percentage of specific lysis of each well on one 48-well plate showing > 10% specific lysis. Overall, all of the peptides tested induced a specific CTL response in vitro on human PBLs, with peptide KLPDLCTEL showing the strongest and peptide FAFKDLFVV the weakest response, as seen by the average percentage of lysis of each assay. The four other peptides, LQDIEITCV, ELTEVFEFA, KTVLELTEF, and TLQDIVLHL, induced an intermediate average lyric activity in vitro. In each assay, a HBc protein- derived, HLA-A*0201-restricted peptide was included as a positive control (FLPSDYFPSV), which induced an average lytic activity of 25.2% specific lysis.




The HBc peptide served as a reference for estimation of precursor frequencies because it is known to be immunogenic in

vitro as well as in vivo in humans (35). Each well in every assay was treated as a single culture, and therefore we have been able to estimate precursor frequencies for each peptide tested. The precursor frequency pCTL was calculated by the assumption that a positive CTL response in a well of the 48-well plate was derived from a single responding cell in the primary stimulation. The probability of 90% of a monoclonal response in a total amount of 24 • 106 CD8 + T cells in the primary induction would require no more than 0.2-0.3 specific precursor cells/ well (36), reflecting a specific pCTL of 1 in 2.5-1.6 • 106 CD8+ T cells. Therefore, we can conclude that the observed pCTL values are indeed derived from monoclonal responses. We have considered all peptides with a similar or higher frequency than the HBc peptide (1 in 1.6 • 106 CD8+ T cells) to be highly immunogenic for human PBLs of normal donors, however. Two peptides (LQDIEITCV and FAFKDLFVV) did not meet the criteria. Therefore, we considered these two peptides not to have a suitable precursor frequency for vaccinations in human PBLs.

Overall, we have been able to determine the binding affinities of all possible 9-mer peptides in HPV-18 E6 and E7 proteins and to rank these peptides according to their affinities to human HLA-A molecules. Moreover, we identified five peptides derived from E6 and one peptide derived from E7 to be immunogenic in vitro in HLA-A*0201-positive PBLs to various degrees, as well as determined the precursor frequencies of the peptide-specific CTLs in PBLs of healthy donors. Taken together, we conclude that the peptides ELTEVFEFA, KLPDLCTEL, and KTVLELTEV of E6 and TLQDIVLHL of E7 are good candidates for the development of a vaccine because of their immunogenicity as well as precursor frequencies in PBLs of healthy donors.

D i s c u s s i o n

Here we report for the first time binding affinities for 9-mer peptides derived from HPV-18 E6 and E7 transforming proteins to purified HLA-A molecules as well as immunogenicity of selected peptides in vitro on human PBLs. Similar results have been reported earlier by Yoon et al. (28) in 1998 for peptides derived from HPV-18 E6 protein binding to the HLA-A*0201 molecule. In contrast to this earlier publication, we not only analyzed and identified high-affinity binding peptides derived from the E6 protein to HLA-A*0201 but also identified and determined binding affinities for E7-derived peptides. More- over, we also included four other, major HLA molecules, HLA- A*0101, HLA-A*0302, HLA-A*ll01, and HLA-A*2402, in our analysis and thereby obtained a complete set of data for each possible 9-mer peptide derived from E6 and E7 proteins in correlation with its affinity to the five major HLA-A molecules. Overall, our data should allow the rapid identification of high- affinity binding peptides to the five major HLA-A molecules and limit the number of peptides to be tested for immunogenicity of the five major HLA-A haplotypes.

Several publications reported a direct correlation between binding affinity and immunogenicity (33, 34), and therefore it is reasonable to focus on peptides with a high binding affinity to

the HLA molecules. Our method allowed us to rank the peptides according to their affinities and therefore identify candidate peptides with high affinities for subsequent testing for immunogenicity. Additionally, computer algorithms for the prediction of potential MHC class I and class II binding peptides for a wide range of HLA and H-2 haplotypes are easily available via the Internet (21-23). Nevertheless, the high affinity binding peptides were often not within the top five peptides, and not all high-affinity binding peptides were actually listed and identified by the computer-based algorithms, e.g., by analyzing the amino acid sequence of HPV-18 E6 protein by the SYFPEITHI computer algorithm for HLA-A*0201 (23). The peptide ELTEVFEFA appears only at position 40 in the ranking of the possible binding peptides, and to cover the four high-affinity binding peptides (KLPDLCTEL, LQDIEITCV, KTVLELTEV, and FAFKDLFVV), the first 18 peptides of the score list would have to be tested. Additionally, not all of the predicted peptides actually bind the HLA-A molecule, e.g., Yoon et al. (28) reported that from initially 18 peptides identified by the computer algorithm for binding to HLA-A*0201, only 8 were actually binding to HLA-A*0201 expressed on T2 cells. Our data will help these algorithms to improve over time and their prediction to become more reliable in the future.

The binding of a peptide to a HLA-A molecule does not necessarily induce a CTL response, and therefore all of the identified high-affinity peptides were tested in vitro for their ability to induce human T-cell responses in vitro. Because the HLA-A*0201 molecule is the most prominent molecule in the human population, we focused on identification of immunogenic, HLA-A*0201-restricted peptides. As a control peptide, we chose the HBc protein-derived immunogenic peptide FLPS- DYFPSV, which induces a primary immune response in vitro

with PBLs from normal donors, over the more commonly used influenza peptide, which induces a recall response for most donors. This approach allowed us to compare the induced primary responses in vitro fi'om our HPV-18 peptides to the HBc peptide of which precursor frequencies in normal donors were published earlier (1 in 1.6 • 106 CD8+ T cells; Ref. 35). Additionally, this peptide has been shown to induce immune responses in humans upon vaccination, and therefore we considered this peptide a more accurate control than the influenza matrix peptide. Additionally, we chose to work with purified CD8 + T cells rather than bulk cultures, allowing us the direct estimation of precursor frequencies of peptide-specific T cells. Because primary immune responses are critically dependent on optimal and prolonged stimulation by antigen-presenting cells (37), we decided to use DCs as the primary antigen-presenting cells in vitro. Dendritic cells have been reported to be the most potent stimulator cells for CD8 + T cells and to induce primary peptide-specific CTL responses with low precursor frequencies in vitro (38-40). In these in vitro immunization assays, the activation status of the DCs is of utmost importance, and im- mature or low-level activated, peptide-loaded DCs do not induce detectable CTL responses in vitro (data not shown), 4 whereas activated, peptide-loaded DCs are excellent inducers and acti-

4 Unpublished results.




vators of peptide-specific CTL responses in vitro with purified CD8 + T cells. The protocol used for production of DCs gen- erates fully activated DCs with high surface expression of MHC class I and class II molecules as well as costimulatory molecules CD80 and CD86 (as determined by FACS analysis). In particular, the maturation step in conditioned medium induces an up-regulation of CD80 expression on DCs, and therefore it is an essential step to be able to activate and induce proliferation of peptide-specific precursor CTLs by peptide-loaded DCs. We have been able use the protocol to determine the immunogenicity of HLA-A*0201 binding peptide from HPV-18 E6 and E7 proteins as well as to determine the precursor frequencies for the immunogenic peptides. Overall, all peptides gave detectable CTL responses. Nevertheless, the peptide FAFKDLVV gave only weak responses in three of the four donors tested and did not induce a detectable CTL response in one donor. In contrast, the peptide KLPDLCTEL induced a strong cytolytic activity in all four donors. Overall, we determined the peptide KLPDLCTEL to be highly immunogenic; peptides LQDIE|TCV, ELTEVFEFA, and KTVLELTEV of E6 as well as TLQDIVLHL of E7 to be intermediately immunogenic; and peptide FAFKDLFVV to be only weakly immunogenic. Sur- prisingly, this is the peptide with the highest affinity for the HLA-A*0201 molecule; the three E6-derived peptides all had intermediate affinities for the HLA-A*0201 molecule. The same result is also reflected in the precursor frequencies as determined by the number of wells/plate showing a positive response. Peptide FAFKDLFVV as well as peptide LQDIETICV had a much lower precursor frequency in normal donors as compared with the other E6-derived peptides. Peptide ELTEVFEFA showed an identical high precursor frequency as the control peptide HBc as well as a comparable immunogenicity. There- fore, it can be concluded that based on the precursor frequencies the peptide ELTEVFEFA (derived from E6) is a highly immunogenic peptide and a good candidate for a vaccine development; peptides KTVLELTEV and KLPDLCTEL (E6 derived) as well as peptide TLQDIVLHL (derived from E7) are weakly immunogenic, and peptides FAFKDLFVV and LQDIETICV may not be suitable candidates for a vaccine development.

Yoon et al. (28) included also results from in vitro immunization assays with the E6-derived peptides in their publication in 1998. Although results of their study are comparable with our results, the authors did not determine peptide KTVLELTEV to be immunogenic. Yoon et al. (28) analyzed in vitro immunization cultures from tour donors tested with peptide KTVLELTEV and found none of the four donors to respond to the peptide in vitro. In contrast, we identified the peptide KTVLELTEV to be immunogenic in all four donors tested. This difference could be explained by the different in vitro immunization protocols used (bulk culture versus purified CD8 + cells). Moreover, we used activated DCs as antigen-presenting cells, which are very potent inducers of CTL responses compared with PBMCs used by Yoon et al. (28). The limitation of the method used by Yoon et al. (28) becomes evident by comparing the percentage of donors with positive responses to our data. In our hands, all of the tested peptides induce a CTL response in all of the donors (therefore, 100% response rate). Yoon et al. (28) showed a maximum of 40% of donors responding to their best peptide (KLTNTGLYNL) and 0 and 33.3% of donors respond-

ing to the other peptides we present in this article. Therefore, our more laborious approach is more reliable and accurate in deter- mining the immunogenicity of individual peptides.

Overall, we have been able to determine the binding affinities of all possible 9-mer peptides derived from HPV- 18 E6 and E7 proteins to the MHC class I molecules HLA-A*0101, HLA- A*0201, HLA-A*0302, HLA-A*ll01, and HLA-A*2402101. Moreover, we have identified the immunogenicity and CTL precursor frequencies of 6 HLA-A*0201-restricted peptides by in vitro immunization assays with human PBL. Taken together, we conclude that the peptides ELTEVFEFA, KLPDLCTEL, KTVLELTEV, and TLQDIVLHL are good candidates for the development of a vaccine because of their immunogenicity as well as precursor frequencies in PBLs of healthy donors. An- other plus point of the data are that the pCTL frequencies provide a baseline for future vaccination studies, e.g., in future clinical trials peptide-specific responses could be monitored by FACS with peptide-loaded tetramers or direct sorting of CTLs from patients (41), thereby circumventing the laborious culture methods by allowing a direct staining of specific T cells.

The final step in our analysis would be to determine which of the identified peptides is endogenously processed and presented by tumor cells. Of particular interest is peptide KLPDLCTEL, because a similar peptide, KLPDQCTEL, exists in HPV-16 E6 protein. The KLPDQCTEL peptide by itself is not immunogenic in vitro (data not shown), but there is some evidence that the same peptide might be endogenously processed and presented by HLA-A*0201, as shown in transfection experiments with a modified vaccinia virus containing the E6

gene of HPV-16 (42). If the KLPDLCTEL peptide of HPV-18 is immunogenic and specific CTLs would cross-react with the HPV-16 peptide, a vaccine containing the KLPDLCTEL peptide would be suitable for prevention or treatment of both HPV types HPV-16 as well as HPV-18.

Acknowledgments We thank Dr. W. Basaam for help with peptide synthesis as well as

P. Simms for excellent help and assistance with FACS analysis.

References 1. Bosch, F. X., Manos, M. M., Munoz, N., Sherman, M., Jansen, A. M., Peto, J., Schiffinan, M. H., Moreno, V., Kurman, R., and Shah, K. V. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. J. Natl. Cancer Inst., 87: 796-801, 1995. 2. Kotloff, K. L., Wasserman, S. S., Russ, K., Shapiro, S., Daniel, R., Brown, W., Frost, A., Tabara, S. O., and Shah, K. Detection of genital human papillomavirus and associated cytological abnormalities among college women. Sex Transm. Dis., 25: 243-250, 1998. 3. Kast, W. M., Feltkamp, M. C. W., Ressing, M. E., Vierboom, M. P. M., Brandt, R. M. P., and Melief, C. J. M. Cellular immunity against human papillomavirus associated cervical cancer. Semin. Virol., 7:117-123, 1996. 4. Koutsky, L. Epidemiology of genital human papillomavirus infection. Am. J. Med., 102: 3-8, 1997. 5. Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J., Peto, J., Meijer, C. J., Munoz, N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol., 189: 12-19, 1999. 6. Lorincz, A. T., Reid, R., Jenson, A. B., Greenberg, M. D., Lancaster, W., and Kurman, R. J. Human papillomavirus infection of the cervix:




relative risk associations of 15 common anogenital types. Obstet. Gy- necol., 79: 328-337, 1992.

7. Burger, R. A., Monk, B. J., Kurosaki, T., Anton-Culver, H., Vasilev, S. A., Berman, M. L., and Wilczynski, S. P. Human papillomavirus type 18: association with poor prognosis in early stage cervical cancer. J. Natl. Cancer Inst., 88: 1361-1368, 1996.

8. Melief, C. J. M., and Kast, W. M. Potential immunogenicity of oncogene and tumor suppressor gene products. Curr. Opin. Immunol., 5: 709-7t3, 1993.

9. Nakagawa, M., Stites, D. P., Farhat, S., Sisler, J. R., Moss, B., Kong, F., Moscicki, A. B., and Palefsky, J. M. Cytotoxic T lymphocyte responses to E6 and E7 proteins of human papillomavirus type 16: relationship to cervical intraepithelial neoplasia. J. Infect. Dis., 175: 927-931, 1996.

10. Ressing, M. E., van Driel, W. J., Celis, E., Sette, A., Brandt, M. P., Hartman, M., Anholts, J. D., Schreuder, G. M., ter Harmsel, W. B., Fleuren, G. J., Trimbos, B. J., Kast, W. M., and Melief, C. J. Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen--A*0201-restricted E7-encoded epitope. Cancer Res., 56: 582- 588, 1996. 11. Nimako, M., Fiander, A. N., Wilkinson, G. W., Borysiewicz, L. K., and Man S. Human papillomavirus-specific cytotoxic T lymphocytes in patients with cervical intraepithelial neoplasia grade III. Cancer Res., 57: 4855-4861, 1997. 12. Feltkamp, M. C. W., Smits, H. L., Vierboom, M. P. M., Minnaar, R. P., de Jongh, M., Drijfhout, J. W., Ter Schegget, J., Melief, C. J. M., and Kast, W. M. Vaccination with cytotoxic T lymphocyte epitope- containing peptide protects against a tumor induced by human Papillo- mavirus type 16-transformed cells. Eur. J. Immunol., 23: 2242-2246, 1993. 13. Feltkamp, M. C., Vreugdenhil, G. R., Vierboom, M. P., Ras, E., Van den Burg, S. H., Ter Schegget, J., Melief, C. J. M., and Kast, W. M. Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16 induced tumors. Eur. J. Immunol., 25: 2638-2642, 1995.

14. Mayordomo, J. I., Zorina, T., Storkus, W. J., Celluzi, C., Falo, L. D., Melief, C. J. M., Ilstad, S. T., Kast, W. M., De Leo, A., and Lotze, M. T. Bone-marrow derived dendritic cells pulsed with tumor peptides effectively treat established murine tumors. Nat. Med., 1: 1297-1302, 1995. 15. Velders, M. P., Nieland, J. D., Rudolf, M. P., Loviscek, K., Wei- jzen, S., de Visser, K. E., Macedo, F. M., Carbone, M., and Kast, W. M. Identification of peptides for immunotherapy of cancer: it is still worth the effort. Crit. Rev. Immunol., I8: 7-27, 1998. 16. Bona, C., Casares, S., and Brumeanu, T. D. Towards development of T-cell vaccines. Immunol. Today, I9: 126-132, 1998.

17. Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med., 4: 328-332, 1998. 18. van Driel, W. J., Ressing, M. E., Kenter, G. G., Brandt, R. M., Krul, E. J., van Rossum, A. B., Schuuring, E., Offringa, R., Bauknecht, T., Tamm-Hermelink, A., van Dam, P. A., Fleuren, G. J., Kast, W. M., Melief, C. J., and Trimbos, J. B. Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a Phase I-II trial. Eur J Cancer, 35: 946-952, 1999. 19. Romero, P., and Cerottini, J. C. Novel methods to monitor antigen- specific cytotoxic T-cell responses in cancer immunotherapy. Mol. Med. Today, 7: 305-312, 1998. 20. Ressing, M. E., van Driel, W. J., Brandt, R. M., Kenter, G. G., de Jong, J. H., Bauknecht, T., Fleuren, G. J., Hoogerhout, P., Offringa, R., Sette, A., Celis, E., Grey, H., Trimbos, B. J., Kast, W. M., and Melief, C. J. Detection of T helper responses, but not of human papillomavirus- specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma. J. Immunother., 23: 255-266, 2000.

21. D'Amaro, J., Houbiers, J. G., Drijfhout, J. W., Brandt, R. M., Schipper, R., Bavinck, J. N., Melief, C. J., and Kast, W. M. A computer

program for predicting possible cytotoxic T lymphocyte epitopes based on HLA class I peptide-binding motifs. Hum. Immunol., 43: 13-18, 1995.

22. Parker, K. C., Bednarek, M. A., and Coligan, J. E. Scheme for ranking potential HLA-A*0201 binding peptides based on independent binding of individual peptide side-chains. J. Immunol., 152: 163-166, 1994.

23. Rammensee, H. G., Bachmann, J., Emmerich, N., and Stevanovic, S. SYFPEITHI. An internet database for MHC tigands and peptide motifs (access via: http://www.uni-tuebingen.de/uni/kxi/).

24. Ressing, M. E., Sette, A., Brandt, R. M. P., Ruppert, J., Wentworth, P. A., Hartman, M., Oseroff, C., Grey, H. M., Melief, C. J. M., and Kast, W. M. Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 proteins identified through in vivo and in vitro immunogenicity studies of HLA-A*0201 binding peptides. J. Immunol., 154: 5934-5943, 1995.

25. Celis, E., Tasi, V., Crimi, C., DeMars, R., Wentworth, P. A., Chesnut, R. W., Grey, H. M., Sette, A., and Serra, H. M. Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes. Proc. Natl. Acad. Sci. USA, 91: 2105-2109, 1994.

26. Wentworth, P. A., Cells, E., Crimi, C., Stitely, S., Hale, L., Tsai, V., Sena, H. M., Del Guercio, M. F., Livingston, B., Alazard, D., Fikes, J., Kubo, R. T., Grey, H., Chesnut, R. W., Chisari, F., and Sette, A. In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides. Mol. hn- munol., 32: 603-612, 1995.

27. Garcia, A. M., Ortiz-Navarrete, V. F., Mora-Garcia, M. L., Flores- Borja, F., Diaz-Quinonez, A., Isibasi-Arattjo, A., Trejo-Becerril, C., Chacon-Salinas, R., Hernandez-Montes, J., Granados-Arreola, J., de Leo, C., and Weiss-Steider, B. Identification of peptides presented by HLA class I molecules on cervical cancer cells with HPV-I 8 infection. Immunol. Lett., 67: 167-177, 1999.

28. Yoon, H., Chung, M. K., Min, S. S., Lee, H. G., Yoo, W. D., Chung, K. T., Jung, N. P., and Park, S. N. Synthetic peptides of human papillomavirus type 18 E6 harboring HLA-A*0201.1 motif can induce peptide-specific cytotoxic T-cells from peripheral blood mononuclear cells of healthy donors. Virus Res., 54: 23-29, 1998.

29. Buus, S., Sette, A., Colon, S. M., Miles, C., and Grey, H. M. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science (Wash- ington DC), 235: 1353-1358, 1987.

30. Kast, W. M., Brandt, R. M., Sidney, J., Drijfhout, J. W., Kubo, R. T., Grey, H. M., Melief, C. J., and Sette, A. Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins. J. Immunol., I52: 3904-3912, 1994.

31. Sette, A., Southwood, S., O'Sullivan, D., Gaeta, F. C., Sidney, J., and Grey, H. M. Effect of pH on MHC class II-peptide interactions. J. Immunol., 148: 844-851, 1992.

32. Romani, N., Reider, D., Heuer, M., Ebner, S., Kampgen, E., Eibl, B., Niederwieser, D., and Schuler, G. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J. Immunol. Methods, 196: 137-151, 1996.

33. Feltkamp, M. C., Vierboom, M. P., Kast, W. M., and Melief, C. J. Efficient MHC class I-peptide binding is required but does not ensure MHC class 1-restricted immunogenicity. Mol. Immunol., 31: 1391- 1401, 1994.

34. Sette, A., Vitiello, A., Reherman, B., Fowler, P., Nayersina, R., Kast, W. M., Melief, C. J., Oseroff, C., Yuan, L., and Ruppert, J. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J. Immunol., 153: 5586-5592, 1994.

35. Livingston, B. D., Crimi, C., Grey, H., Ishioka, G., Chisari, F. V., Fikes, J., Grey, H., Chesnut, R. W., and Sette, A. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those by acute viral infection. J. Immunol., 159: 1383-1392, 1997.




36. Coligan, J. E., Kruisbeck, A. M., Margulies, D. H., Shevach, E. M., and Strober, W. (eds.). Current Protocols in Immunology, National Institutes of Health, pp. 3.15.10. New York: John Wiley and Sons, 1999. 37. Iezzi, G., Karjalainen, K., and Lanzavecchia, A. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity, 8: 89-95, 1998.

38. Zarling, A. L., Johnson, J. G., Hoffman, R. W., and Lee, D. R. Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells. J. Immunol., 162:5197-5204, 1999. 39. Wilson, C. C., Olson, W. C., Tuting, T., Rinaldo, C. R., Lotze, M. T., and Storkus, W. J. HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Thl-biasing cytokines. J. Immu- nol., 162: 3070-3078, 1999. 40. Mortarini, R., Anichini, A., Di Nicola, M., Siena, S., Bregni, M., Belli, F., Molla, A., Gianni, A. M., and Parmiani, G. Autologous

dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent antigen-presenting cells in the induction of melan-A/Mart-l(27-35)-specific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors. Cancer Res., 57: 5534-5541, 1997.

41. Youde, S. J., Dunbar, P. R., Evans, E. M., Fiander, A. N., Bo- rysiewicz, L. K., Cerundolo, V., and Man, S. Use of fluorogenic histocompatibility leukocyte antigen-A*0201/HPV 16 E7 peptide complexes to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous human papillomavirus antigens. Cancer Res., 60:365-371, 2000.

42. Bartholomew, J. S., Stacey, S. N., Coles, B., Burt, D. J., Arrand, J. R., and Stern, P. L. Identification of a naturally processed HLA- A*0201-restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein. Eur. J. Immunol., 24:3175-3179, 1994.



2001;7:788s-795s. Clin Cancer Res Michael P. Rudolf, Stephen Man, Cornelis J. M. Melief, et al. E6 and E7Affinity Peptides of Human Papillomavirus Type 18 Proteins Human T-Cell Responses to HLA-A-restricted High Binding

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