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Immunology |
Department of Immunology, Institute for Cell Biology [T. W., C. G., M. S., F. O., S. W., O. S., S. S., H-G. R.] and Departments of Obstetrics and Gynecology [R. K.], Urology [W. L., K-H. B.], and Transfusion Medicine [D. W.], University of Tübingen, D-72076 Tübingen, Germany
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparative expression profiling of a tumor and the corresponding autologous normal tissue enabled by DNA microarray technology (11 , 12) is an excellent method for identifying large numbers of candidate TAAs from individual tumor samples (13, 14, 15) . HLA-presented peptides from overexpressed or selectively expressed proteins should provide targets for specific CTL recognition of tumors. The feasibility of combining expression analysis with epitope prediction for a successful vaccine design has been demonstrated in a mouse model (16) . However, epitope prediction, even for only a few target genes, results in a vast number of candidate peptides, the majority of which are actually not presented by MHC molecules. A combination of epitope prediction with biochemical verification would be ideal.
Here we describe an integrated functional genomics approach that provides the basis for making an individual selection of peptides that can be used for peptide-based multiepitope immunotherapy tailored to suit the needs of each patient (Fig. 1)
. We analyzed samples from surgically removed malignant and normal tissue and blood from RCC patients in three different ways.
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MATERIALS AND METHODS |
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Isolation of MHC Class I-bound Peptides.
Shock-frozen tumor samples were essentially processed as described previously (10)
. Peptides were isolated according to standard protocols (19)
using the HLA class I-specific mAb W6/32 (20)
or HLA-A2-specific mAb BB7.2 (21)
.
MS.
Peptides from tumor RCC01 were separated by reversed-phase high-performance liquid chromatography (SMART system, µRPC C2/C18 SC 2.1/10; Amersham Pharmacia Biotech, Freiburg, Germany), and fractions were analyzed by nano-ESI MS on a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF; Micromass, Manchester, United Kingdom) as described previously (10)
. Peptides from tumor RCC13 were identified by on-line capillary LC-MS as described previously (10)
, with minor modifications. Sample volumes of about 100 µl were loaded, desalted, and preconcentrated on a 300 µm x 5-mm C-18 µ-Precolumn (LC Packings, San Francisco, CA). A syringe pump (PHD 2000; Harvard Apparatus, Inc., Holliston, MA) equipped with a gas-tight 100-µl syringe (1710 RNR; Hamilton, Bonaduz, Switzerland) delivered solvent and sample at 2 µl/min. For peptide separation, the preconcentration column was switched in line with a 75 µm x 250-mm C-18 column (LC Packings). A binary gradient of 25–60% B within 70 min was performed, applying a 12 µl/min flow rate reduced to approximately 300 nl/min with a precolumn split using a TEE piece (ZT1C; Valco, Schenkon, Switzerland) and a 300 µm x 150 mm C-18 column as a backpressure device. A blank run was always included to ensure that the system was free of residual peptide. On-line fragmentation was performed as described previously (10)
. Fragment spectra were analyzed manually, and database searches (NCBInr, EST) were made using MASCOT4
(22)
.
Preparation of RNA.
Fragments of normal and malignant renal tissue were dissected, shock-frozen, ground by mortar and pestle under liquid nitrogen, and homogenized with a rotary homogenizer (Heidolph Instruments, Schwabach, Germany) in Trizol (Life Technologies, Inc., Karlsruhe, Germany). Total RNA was prepared according to the manufacturer’s protocol followed by a clean-up with RNeasy (Qiagen, Hilden, Germany). Total RNA from human tissues was obtained commercially (human total RNA Master Panel II Clontech, Heidelberg, Germany).
High-density Oligonucleotide Microarray Analysis.
Double-stranded DNA was synthesized from 40 µg of total RNA using SuperScript RTII (Life Technologies, Inc.) and the primer (Eurogentec, Seraing, Belgium) designated by the Affymetrix manual. In vitro transcription using the BioArray High Yield RNA Transcript Labeling Kit (ENZO Diagnostics, Inc., Farmingdale, NY), fragmentation, hybridization on Affymetrix HuGeneFL GeneChips (Affymetrix, Santa Clara, CA), and staining with streptavidin-PE and biotinylated anti-streptavidin antibody (Molecular Probes, Leiden, The Netherlands) followed the manufacturer’s protocols (Affymetrix). The Affymetrix GeneArray Scanner was used, and data were analyzed with Microarray Analysis Suite 4.0 software. After scaling, exclusive expression was determined by the absolute call algorithms. Only genes showing an increase according to the difference call algorithm were considered to be up-regulated by the factor as given by the fold change, calculated from average difference changes.
Real-Time RT-PCR.
The cDNA generated for microarray analysis was used for qPCR analysis. Tissue expression of adipophilin and keratin 18 was analyzed using single-stranded cDNA synthesized from 1 µg of total RNA by SuperScript RTII (Life Technologies, Inc.) and a random hexamer primer. Each gene was run in duplicates (40 cycles of 95°C for 15 s and 60°C for 1 min) using SYBRGreen chemistry on the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). Samples were independently analyzed two to three times. Primers (MWG-Biotech, Ebersberg, Germany) were selected to flank an intron, and PCR efficiencies were tested for all primer pairs and found to be close to 1. Primer sequences (forward and reverse) were as follows: (a) ADFP, 5'-CACTGTGCTGAGCAATTTGAG-3' and 5'-TTGGCTTGATCTTGGATGTTC-3'; (b) KIAA0367, 5'-AATGCCTCAGTAGTTTGTTCCC-3' and 5'-TTTATTCTGAGCAATCCAATGC-3'; (c) LGALS2, 5'-AAGATCACAGGCAGCATCG-3' and 5'-GACAATGGTGGATTCGCTG-3'; (d) CCND1, 5'-CACGATTTCATTGAACACTTCC-3' and 5'-TGAACTTCACATCTGTGGCAC-3'; (e) MET, 5'-ACATTGAAATGCACAGTTGGTC-3' and 5'-ACAGGATCCACATAGGAGAATG-3'; (f) ETS1, 5'-AAAGTGCCAACTTCCCCTG-3' and 5'-GGAAATCCGACTTTCTTCCC-3'; (g) KRT18, 5'-GAGCCTGGAGACCGAGAAC-3' and 5'-TTGCGAAGATCTGAGCCC-3'; (h) LMP2, 5'-TGGGATAGAACTGGAGGAACC-3' and 5'-CATATACCTGACCTCCTTCACG-3'; (i) LMP7, 5'-CTATCTGCGAAATGGAGAACG-3' and 5'-CCTTCTTATCCCAGCCACAG-3'; and (j) 18S rRNA, 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3'. PCR products were analyzed on 3% agarose gels for purity and sequence-verified after cloning into pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Groningen, the Netherlands). Data analysis involved the 
CT method for relative quantification.
LCM.
Embedded frozen-tissue specimens were cut at 6-µm thickness and transferred in 70% ethanol for less than 15 min. Slides were incubated for 90 s in Mayer’s hematoxylin (Merck, Darmstadt, Germany); rinsed in water; and incubated for 1 min in 70% ethanol, 1 min in 95% ethanol, 30 s in 1% alcoholic eosin Y (Sigma, Munich, Germany), 2 x 2 min in 95% ethanol, 2 x 2 min in 100% ethanol, and, finally, 2 x 5 min in xylene. After air-drying for 15 min, slides were stored under dry conditions. Nonmalignant epithelial tubular cells and carcinoma cells were isolated by LCM using the PixCell II LCM System (Arcturus Engineering, Harpenden, United Kingdom). Total RNA was extracted in 400 µl of Trizol.
PBMCs, Tetramer Production, and Flow Cytometry.
Buffy coats from HD were provided by the Bloodbank (Tübingen, Germany). HD1 and HD2 were serologically typed as CMV positive, and HD4 and HD6 were serologically typed as CMV negative. PBMCs were isolated by gradient centrifugation (FicoLite H, Wertheim-Bettingen, Germany) and frozen. HLA-A*0201 tetrameric complexes were produced as described previously (18
, 23)
, with minor modifications. The HLA-A2 binding peptides used for the refolding were ALLNIKVKL from keratin 18 and the epitope NLVPMVATV from pp65 HCMVA. Tetramers were assembled by mixing biotinylated monomers with streptavidin-PE or streptavidin-APC (Molecular Probes) at a 4:1 ratio. PBMCs were thawed, and 2–3 x 106 cells were incubated for 30 min at 4°C with both tetramers (10 µg/ml for each monomer in PBS, 0.01% NaN3, 2 mM EDTA, and 50% FCS; optimal concentration determined after titration experiments), and then anti-CD4-FITC (Coulter-Immunotech, Hamburg, Germany) and anti-CD8-PerCP (Becton Dickinson, Heidelberg, Germany) mAbs were added for 20 min. After three washes, samples were fixed in FACS buffer and 1% formaldehyde. Four-color analysis was performed on a FACSCalibur cytometer (Becton Dickinson). Under these conditions, the average background of nonspecific staining with tetramers was found to be <0.01% on CD8+ cells.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Most of the overexpressed genes might be cancer related, i.e., they are either oncogenes, tumor suppressor genes, or genes already described as overexpressed in cancer, such as cyclin D1 (CCND1, increased by factor 4.9; data not shown; Ref. 24
), carbonic anhydrase IX (CA9; Ref. 25
), cerebroside sulfotransferase (CST; Ref. 26
), and parathyroid hormone-like hormone (Ref. 27
; Table 1
). The cancer-associated adipose differentiation-related protein (ADFP), or adipophilin, showed the second-highest degree of overexpression. We compared the list of overexpressed genes with the SEREX database5
(28)
and found KIAA0367 to be included (clone ID NGO-St-87). Both tumors showed increased expression levels of IFN-
-inducible genes: TAP1, MHC class I heavy chain, and the immunoproteasomal subunits LMP2 and LMP7 (data not shown).
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. Overexpression of adipophilin (ADFP) and cyclin D1 (CCND1) and equal expression of ets-1 (ETS1) was confirmed. Relative expression levels detected by both techniques were roughly comparable: for example, adipophilin was overexpressed in RCC01 by a factor of 29.1 by microarray, compared with 18.1 by qPCR. The corresponding numbers in RCC13 are 11.4 and 6.7 (Fig. 2A)
. Galectin 2 (LGALS2) was overexpressed in RCC01, and keratin 18 (KRT18) was overexpressed in RCC13, but not vice versa. An exception to the congruence between microarray and qPCR was the overexpression of KIAA0367 and met proto-oncogene (MET) in RCC13. Analysis of pure tumor and normal cell populations obtained by LCM revealed the expression of adipophilin and galectin 2 in the tumors of both patients, but not in normal tissue (data not shown). To confirm that LMP2 and LMP7 are indeed up-regulated in tumor cells, we also performed qPCR on LCM-derived cells (Fig. 2B)
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. To our knowledge, this is the largest number of ligands identified from a single solid tumor.
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). Twenty-one of 25 identified HLA-A*02-restricted ligands were new; 4 have been reported previously. Most ligands were derived from abundantly expressed housekeeping proteins, but we could also detect ligands from proteins with reported tumor association such as YVDPVITSI, derived from met proto-oncogene, ALLNIKVKL from keratin 18, and SVASTITGV from adipophilin. HLA-A*68 ligands were recognized by their anchor amino acids T/I/V/A/L in position 2 and COOH-terminal R/K, which also indicated that the subtype was most probably HLA-A*6801. Two other ligands from adipophilin, MTSALPIIQK and MAGDIYSVFR, were found among HLA-A*68-presented peptides, as well as ETIPLTAEKL, derived from tumor-associated cyclin D1. Annexin II, from which the peptide TIVNILTNR was identified, has been shown to be immunogenic in the context of MHC class II in melanoma patients (29)
.
All other 35 ligands carried E in position 2, an anchor residue of HLA-B*44. The peptide motif of HLA-B*18 is still unknown; therefore, a distinction between ligands of these two HLA-B molecules was not possible. Fragments of vimentin (EEIAFLKKL) and caldesmon (DEAAFLERL), both overexpressed in RCC (13)
, were identified as well as ligands derived from ets-1 (NEFSLKGVDF), 
-catenin (NEQDLGIQY), and galectin 2 (SEVKFTVTF).
Comparison with microarray data indicated 10 overexpressed genes as sources of MHC ligands: adipophilin, KIAA0367, SEC14-like 1, B-cell translocation gene 1, aldolase A, cyclin D1, annexin A4, catenin 1, galectin 2, and LMP2. Three of them were also included in the SEREX database: KIAA0367, aldolase A, and catenin 1.
Highest Expression Levels of Adipophilin in Tumors Compared with the Most Essential Human Organs and Tissues.
If the difference in expression of a particular antigen between tumor cells and all normal cells is high, this antigen should also have greater potential as a possible vaccine candidate. To this end, we analyzed the expression of adipophilin and keratin 18 in 21 human tissues and organs in addition to the kidney. Adipophilin, which was selectively overexpressed in the two tumors tested for comparison, was only marginally expressed in all other organs. Kidney was the organ with the highest expression level of adipophilin among all analyzed normal tissues (Fig. 3A)
. In contrast to adipophilin, keratin 18 showed a more heterogeneous tissue distribution, in particular with high expression levels in colon and placenta (Fig. 3B)
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and keratin 18 (data not shown). Interestingly, the keratin 18-derived ligand was also presented on a solid colon carcinoma (data not shown). In addition, several other ligands were identified from RCC13 (Table 2)
. We found one ligand derived from nicotinamide N-methyltransferase, a gene that is overexpressed in >95% of all RCCs (14)
. Some other ligands overlap with the peptide repertoire of RCC01.
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Keratin 18-specific T Cells Are Present in the Normal CD8+ T-Cell Repertoire.
PBMCs from patients RCC01 and RCC13 were not available, which prevented us from testing T-cell reactivity against the frameshift-derived peptide identified above. Nevertheless, we tested six HLA-A2-positive RCC patients for reaction against four of the relevant peptides (HLA-A*02-restricted ligands from adipophilin, keratin 18, KIAA0367, and met proto-oncogene; see Table 2
). We used a very sensitive qPCR assay for IFN- mRNA production by CD8+ T cells (17)
, following a 7-day in vitro sensitization with peptide. In all patients, high production of IFN- mRNA was observed after stimulation with recall viral-derived peptides, showing that the cells were functional after isolation and stimulation. In contrast, responses against KIAA0367 were not detected, and sporadic but marginal responses were seen after stimulation with met proto-oncogene, keratin 18, or adipophilin peptides (data not shown). We also stained PBMCs of few patients and healthy individuals with HLA-A*0201 tetramers folded with either the adipophilin, keratin 18, or met proto-oncogene peptides. A significant population of CD8+ T lymphocytes specific for keratin 18 (between 0.02% and 0.2% of CD8+ T cells) was found in 4 of 22 healthy individuals tested (Fig. 5
shows the staining of two positive and two negative donors; 0.02% of CD8+ T cells for HD1 and 0.17% of CD8+ T cells for HD6 bind to the keratin 18 tetramer). These results were reproduced in at least three independent experiments using two or more blood samples obtained at different time points and involving different batches of independently folded keratin 18 tetramers. Moreover, in a colabeling experiment it was shown that the binding of keratin 18 tetramer was specific because this population did not stain with a CMV tetramer (Fig. 5
, bottom row). We conclude that for the keratin 18 peptide at least, specific CD8+ T lymphocytes are contained in the human T-cell repertoire. However, the four peptides identified on RCC are generally not recognized by spontaneously arising T cells detectable in the blood of patients using tetramers ex vivo or even qPCR after one in vitro stimulation with peptides. However, vaccination might lead to activation and expansion of specific CD8+ T cells against these peptides, which then could be detected by accurate T-cell monitoring.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apart from these more obvious points, additional considerations can be made, such as known cancer association, involvement in the oncogenic process, or immunogenicity of the gene product. The analysis of existing databases should support this endeavor. For example, the SEREX database indicates that antibodies against -catenin have been found in renal cancer patients, antibodies against KIAA0367 have been found in stomach cancer patients, and antibodies against vimentin have been found in pancreatic cancer patients, suggesting a spontaneous immune response.
Based on the following detailed discussion of each new ligand, we would propose an individual combination of peptides for vaccination of patients RCC01 (Table 3)
and RCC13 (Table 4)
. Adipophilin is an interesting candidate for a vaccine component for several reasons: first, it is only expressed in a few cell types (31)
such as adipocytes, lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells, and pathological hepatocytes in alcoholic liver cirrhosis. Second, this gene was reported to be overexpressed in cancer (13
, 32)
. Third, we demonstrated that adipophilin is selectively expressed in the tumor cells of RCC01 and RCC13 but not in LCM-derived pure normal kidney cell populations. We also found that expression in all analyzed normal tissues is much lower than in tumor cells (Fig. 3A)
. Finally, we identified three MHC class I ligands from this protein. KIAA0367 was overexpressed in patient RCC01 and according to the SAGEmap,6
a public database for gene expression (33)
, KIAA0367 is mainly expressed in prostate carcinoma and in the brain. Expression analysis by qPCR also showed that the highest levels were found in the brain and prostate (data not shown). Because the brain expresses low MHC and is partially immunoprivileged, and because patient RCC01 was female and thus lacking prostate, the KIAA0367 peptide ALFDGDPHL would seem to be a first-class choice of vaccine component for this patient. According to analysis of LCM-derived samples, galectin 2, the source protein of SEVKFTVTF, was selectively expressed in both tumors and not in normal kidney cells. Other members of the galectin gene family, including galectin 1, galectin 3, and galectin 12, are overexpressed in malignancies (13
, 34)
. Cyclin D1 is also overexpressed in several malignancies, including RCC (24)
. We found an overexpression of cyclin D1 in RCC01, from which ETIPLTAEKL has been eluted, as well as in RCC13. The expression of cyclin D1 in all analyzed healthy organs and tissues was lower compared with RCC01 and RCC13 (data not shown).
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. Thus, this gene is an interesting vaccine candidate for RCC13, especially because of its oncogenic property. Expression analysis in healthy organs and tissues showed the highest level in normal kidney followed by brain and trachea (data not shown). Nicotinamide N-methyltransferase was exclusively expressed in RCC13 compared with the corresponding normal tissue, and expression in other tissues was limited to liver, which still expressed lower amounts compared with the tumor (data not shown). It is highly probable that the frameshift-derived ligand was presented exclusively on tumor cells and for this reason should be included in the vaccine. We identified ligands from keratin 18 and from vimentin, ALLNIKVKL and EEIAFLKKL, respectively. Both genes were overexpressed in RCC13 but not in RCC01. Keratin 18 has been shown to be overexpressed in carcinomas, and the coexpression of keratin 18 and keratin 8 together with vimentin augments tumor cell motility (35 , 36) . However, because of the rather high expression of keratin 18 in colon, we would not recommend the keratin 18-derived ligand for vaccination. We also found MHC class I ligands from three members of the annexin gene family, TIIDILTKR derived from annexin I, TIVNILTNR derived from annexin II, and DEVKFLTV derived from annexin IV. Annexin II-specific CD4+ T cells were reported to specifically recognize melanoma cells overexpressing annexin II (29) , but we did not find overexpression in RCC01, from which this ligand has been eluted. In RCC01, annexin IV was overexpressed, and a ligand from this protein was eluted. However, because annexin IV is mainly expressed in all epithelial cells (37) , this ligand is excluded from the proposed vaccine. All other new ligands derived from overexpressed genes and not discussed in detail (SEC14L1, BTG1, ALDOA, CTNNA1, PSMB9, IFI30, and HLA-E) were not considered to be suitable for vaccination because they either displayed a broad tissue expression pattern or the level of overexpression was too low.
As a second source of vaccine components, known epitopes from described TAAs can be included without hesitation in the vaccine provided that they match the patient’s HLA type and the TAAs are expressed in the individual tumor (Tables 3
and 4
). This can be checked by referring to the expression data. Thus, in our microarray data, we looked for expression of known TAAs such as NY-ESO-1, LAGE-1, Hom-Mel-40, SSX2, SSX4, CT7, MAGE-1, SCP1, MUC-1, RAGE, GAGE, survivin, hTERT, RU1, Her-2/neu, carcinoembryonic antigen, WT1, SART3, gp75, PRAME, and CA9. In RCC01, CA9 was expressed exclusively in the tumor (Table 1)
; the vaccine for this particular patient could thus contain the CA9-derived epitope HLSTAFARV (25)
. Similarly, the two known epitopes of cyclin D1 (38)
could also be applied. These three epitopes would have been relevant for RCC13 as well. In addition, the exclusive expression of PRAME in RCC13 became clear. Thus, the four epitopes of PRAME restricted to HLA-A*02 (39)
and the HLA-A*24-restricted epitope (40)
can be classed as suitable for this patient. Finally, our new HLA-A*02-restricted ligands from adipophilin and KIAA0367 eluted from RCC01 should be included in the selection because of the overexpression of these genes in RCC13.
In addition to identified HLA ligands from overexpressed gene products, epitope prediction7
for the relevant HLA alleles combined with proteasomal cleavage prediction8
(41)
can be applied to overexpressed genes (Fig. 1)
. This provides more potential candidates for vaccination that should be screened for presentation on the solid tumor as carried out for selected peptides in tumor RCC13 by on-line LC-MS. The combination of expression profiling and epitope prediction has been suggested (42
, 43)
and reported previously, but only one target gene has been used to predict murine epitopes, and neither presentation of the predicted epitopes on the cell surface nor type of antitumor activity has been shown (16)
.
There is increasing evidence that the peptide pool generated by immunoproteasome differs from that produced by the standard proteasome (44, 45, 46) . Because we could show up-regulation of immunoproteasome subunits in tumor cells, one might expect differences among the MHC ligands presented on tumor versus normal cells.
All of our identified ligands except the frameshift-derived peptide could be self peptides and, as such, could also be presented on the surface of normal cells. If specific T cells against these peptides exist, they might induce autoimmunity once activated by vaccination. In all human vaccination studies published thus far, no indication of autoimmunity has been reported. For example, TAAs such as carcinoembryonic antigen or Her2/neu, which are broadly expressed by epithelial cells, are being targeted without any sign of autoimmunity in ongoing clinical trials. One exception might be T-cell-mediated vitiligo after antigen-specific therapy of melanoma (47) . However, even in cases in which either whole tumor lysate was loaded onto dendritic cells for vaccination or autologous cancer-derived heat shock proteins were used, no evidence for autoreactivity toward normal tissue was observed (2 , 48) . Therefore, we believe that the risk of inducing autoimmunity using our vaccine candidates is no greater than that in these previous instances.
Being aware of the fact that self-protein-derived peptides might be subject to T-cell tolerance, we selected four of our interesting peptides to be screened for specific peripheral CD8+ T cells in patients and HDs: only in the case of keratin 18 was a significant tetramer-positive population seen in 4 of 22 HDs tested. It is likely that such T cells can also be found in cancer patients, and more RCC patients must be screened to evaluate the frequency of this keratin 18-specific CD8+ population. It is not clear whether these T cells specific for a self antigen are tolerized in vivo, and experiments are being carried out to characterize them functionally. For this reason, and considering the high expression of keratin 18 in normal colon, this antigen does not constitute a first choice candidate target for immunotherapy at the present time. The immunogenicity of those antigens shared among tumors from several patients should be tested, for example, in classical in vitro priming experiments (9
, 25)
. Using tetramer staining or qPCR for IFN-, we could generally detect no spontaneous expansion of specific T cells against the peptides tested after one in vitro stimulation, suggesting either that these T cells are not activated in vivo or that their frequency in the blood is too low to be detected in our assays. Here again, the number of RCC patients screened must be increased. However, we do not believe that the spontaneous in vivo response to these peptides in cancer patients is a prerequisite for their use as a vaccine. Indeed, previous research has shown that induction of tumor peptide-specific CD8+ T cells (not detectable before vaccination) can correlate with clinical benefit (5
, 49
, 50)
. Using qPCR, we also observed the induction of MUC1-specific CD8+ T cells in a vaccinated patient simultaneously to metastatic regression (5)
.9
Moreover, most immunization protocols tested until now have used antigens defined by spontaneous immune responses in patients. One could argue that such spontaneous T- or B-cell responses, in most cases, are not efficient enough to destroy the tumor. We hypothesize that redirecting immunity to tumor-associated structures not obviously attacked might result in more efficient responses.
The combination of gene expression analysis and MHC ligand identification allows the selection of several peptide candidates for immunotherapy. This can be performed in an appropriate time frame after surgical intervention to allow rapid subsequent vaccination. Analysis of the T-cell response before and during vaccination, together with clinical monitoring, will help to determine those peptides that induce a beneficial immune response for the patient. Thus, we have demonstrated that it is possible to extract information from a single excised tumor specimen that leads to an optimized design of a multiepitope, peptide-based vaccine directed against the tumor of an individual patient and considering all expressed HLA alleles.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Deutsche Forschungsgemeinschaft (SFB510, Project C7, and Leibnizprogramm), European Union (QLG2-1999-00713), Deutsche Krebshilfe, CTL Immunotherapies, Cancer Research Institute, and the IZKF Tübingen. 
2 To whom requests for reprints should be addressed, at Department of Immunology, Institute for Cell Biology, Auf der Morgenstelle 15, D-72076 Tübingen, Germany. Fax: 49-7071-29-5653; E-mail: rammensee{at}uni-tuebingen.de
3 The abbreviations used are: TAA, tumor-associated antigen; MS, mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; RCC, renal cell carcinoma; qPCR, quantitative PCR; mAb, monoclonal antibody; LCM, laser capture microdissection; HD, healthy donor; PBMC, peripheral blood mononuclear cell; ESI, electrospray ionization; CMV, cytomegalovirus; PE, phycoerythrin; DDX3, DEAD/H box polypeptide 3.
4 http://www.matrixscience.com.
5 http://www.licr.org/SEREX.html.
6 http://www.ncbi.nlm.nih.gov/SAGE.
9 C. Gouttefangeas, unpublished data.
Received 4/22/02. Accepted 8/15/02.
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J. Dengjel, M.-D. Nastke, C. Gouttefangeas, G. Gitsioudis, O. Schoor, F. Altenberend, M. Muller, B. Kramer, A. Missiou, M. Sauter, et al. Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas. Clin. Cancer Res., July 15, 2006; 12(14): 4163 - 4170. [Abstract] [Full Text] [PDF]
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C. S. Schuetz, M. Bonin, S. E. Clare, K. Nieselt, K. Sotlar, M. Walter, T. Fehm, E. Solomayer, O. Riess, D. Wallwiener, et al. Progression-Specific Genes Identified by Expression Profiling of Matched Ductal Carcinomas In situ and Invasive Breast Tumors, Combining Laser Capture Microdissection and Oligonucleotide Microarray Analysis. Cancer Res., May 15, 2006; 66(10): 5278 - 5286. [Abstract] [Full Text] [PDF]
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S. Hofmann, M. Gluckmann, S. Kausche, A. Schmidt, C. Corvey, R. Lichtenfels, C. Huber, C. Albrecht, M. Karas, and W. Herr Rapid and Sensitive Identification of Major Histocompatibility Complex Class I-associated Tumor Peptides by Nano-LC MALDI MS/MS Mol. Cell. Proteomics, December 1, 2005; 4(12): 1888 - 1897. [Abstract] [Full Text] [PDF]
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H. D. Hickman-Miller, W. Bardet, A. Gilb, A. D. Luis, K. W. Jackson, D. I. Watkins, and W. H. Hildebrand Rhesus Macaque MHC Class I Molecules Present HLA-B-Like Peptides J. Immunol., July 1, 2005; 175(1): 367 - 375. [Abstract] [Full Text] [PDF]
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S. Appel, A. Rupf, M. M. Weck, O. Schoor, T. H. Brummendorf, T. Weinschenk, F. Grunebach, and P. Brossart Effects of Imatinib on Monocyte-Derived Dendritic Cells Are Mediated by Inhibition of Nuclear Factor-{kappa}B and Akt Signaling Pathways Clin. Cancer Res., March 1, 2005; 11(5): 1928 - 1940. [Abstract] [Full Text] [PDF]
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H. Sultmann, A. v. Heydebreck, W. Huber, R. Kuner, A. Buness, M. Vogt, B. Gunawan, M. Vingron, L. Fuzesi, and A. Poustka Gene Expression in Kidney Cancer Is Associated with Cytogenetic Abnormalities, Metastasis Formation, and Patient Survival Clin. Cancer Res., January 15, 2005; 11(2): 646 - 655. [Abstract] [Full Text] [PDF]
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A. Dorrschuck, A. Schmidt, E. Schnurer, M. Gluckmann, C. Albrecht, C. Wolfel, V. Lennerz, A. Lifke, C. Di Natale, E. Ranieri, et al. CD8+ cytotoxic T lymphocytes isolated from allogeneic healthy donors recognize HLA class Ia/Ib-associated renal carcinoma antigens with ubiquitous or restricted tissue expression Blood, October 15, 2004; 104(8): 2591 - 2599. [Abstract] [Full Text] [PDF]
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S. Mocellin, E. Wang, M. Panelli, P. Pilati, and F. M. Marincola DNA Array-Based Gene Profiling in Tumor Immunology Clin. Cancer Res., July 15, 2004; 10(14): 4597 - 4606. [Abstract] [Full Text] [PDF]
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K. Schag, S. M. Schmidt, M. R. Muller, T. Weinschenk, S. Appel, M. M. Weck, F. Grunebach, S. Stevanovic, H.-G. Rammensee, and P. Brossart Identification of C-Met Oncogene as a Broadly Expressed Tumor-Associated Antigen Recognized by Cytotoxic T-Lymphocytes Clin. Cancer Res., June 1, 2004; 10(11): 3658 - 3666. [Abstract] [Full Text] [PDF]
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H. D. Hickman, A. D. Luis, R. Buchli, S. R. Few, M. Sathiamurthy, R. S. VanGundy, C. F. Giberson, and W. H. Hildebrand Toward a Definition of Self: Proteomic Evaluation of the Class I Peptide Repertoire J. Immunol., March 1, 2004; 172(5): 2944 - 2952. [Abstract] [Full Text] [PDF]
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M. R. Muller, G. Tsakou, F. Grunebach, S. M. Schmidt, and P. Brossart Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells Blood, March 1, 2004; 103(5): 1763 - 1769. [Abstract] [Full Text] [PDF]
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S. M. Schmidt, K. Schag, M. R. Muller, T. Weinschenk, S. Appel, O. Schoor, M. M. Weck, F. Grunebach, L. Kanz, S. Stevanovic, et al. Induction of Adipophilin-Specific Cytotoxic T Lymphocytes Using a Novel HLA-A2-Binding Peptide That Mediates Tumor Cell Lysis Cancer Res., February 1, 2004; 64(3): 1164 - 1170. [Abstract] [Full Text] [PDF]
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D. Nagorsen, C. Scheibenbogen, F. M. Marincola, A. Letsch, and U. Keilholz Natural T Cell Immunity against Cancer Clin. Cancer Res., October 1, 2003; 9(12): 4296 - 4303. [Abstract] [Full Text] [PDF]
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S. M. Schmidt, K. Schag, M. R. Muller, M. M. Weck, S. Appel, L. Kanz, F. Grunebach, and P. Brossart Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells Blood, July 15, 2003; 102(2): 571 - 576. [Abstract] [Full Text] [PDF]
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A. Admon, E. Barnea, and T. Ziv Tumor Antigens and Proteomics from the Point of View of the Major Histocompatibility Complex Peptides Mol. Cell. Proteomics, June 1, 2003; 2(6): 388 - 398. [Abstract] [Full Text] [PDF]
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, RCC01 tumor; 
, RCC01 normal tissue;
, RCC13 tumor; 
, RCC13 normal tissue). A, expression of potential target genes. Symbols for gene names are adipophilin (ADFP), galectin 2 (LGALS2), cyclin D1 (CCND1), met proto-oncogene/hepatocyte growth factor receptor (MET), ets-1 (ETS1), and keratin 18 (KRT18). B, expression of immunoproteasome subunits LMP2 and LMP7; bars, ±SE.
