Method for identifying immunoreactive peptides

Abstract

The invention relates to a method for identifying immunoreactive peptides. According to said method, a sample of tumorous and corresponding healthy tissue is first provided, the tumor-specific expression profile is subsequently determined and antigenic peptides are isolated from the tumorous tissue and analyzed. The respective data that has been obtained is then matched and peptides are identified on the basis of said data.

Claims

1. A method for identifying antigenic peptides for use to induce a CTL immune response in an individual comprising: a) determining HLA-subtypes of the individual, b) obtaining a tumor sample and a corresponding healthy tissue sample from the individual, c) identifying one or more genes that are overexpressed in the tumor sample relative to the healthy tissue sample using microarray analysis or RT-PCR, d) identifying one or more antigenic peptides bound to the HLA-subtypes in the tumor sample by mass spectrometry, e) selecting only the antigenic peptides from step d), which are encoded by an overexpressed gene from step c), wherein the antigenic peptides are selected from the group consisting of ALLNIKVKL (SEQ ID NO: 1), YVDPVITSI (SEQ ID NO: 3), SVASTITGV (SEQ ID NO: 4), MTSALPIIQK (SEQ ID NO: 5), MAGDIYSVFR (SEQ ID NO: 6), ALAAVVTEV (SEQ ID NO: 7), and GIGSRGDRS (SEQ ID NO: 8), and f) synthesizing said antigenic peptides selected in step e), wherein the CTL immune response is induced in the individual.

2. The method of claim 1, wherein the antigenic peptides selected in step e) are patient-specific.

3. The method of claim 1, wherein the antigenic peptides selected in step e) are generated by a reading-frame shift mutation.

4. The method of claim 1, wherein after step e) a further step is performed, wherein the reactivity of T-lymphocytes is tested against the antigenic peptides selected in step e).

5. The method of claim 2, wherein after step e) a further step is performed, wherein the reactivity of T-lymphocytes is tested against the antigenic peptides selected in step e).

6. The method of claim 3, wherein after step e) a further step is performed, wherein the reactivity of T-lymphocytes is tested against the antigenic peptides selected in step e).

7. The method of claim 4, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or gamma-Interferon mRNA synthesized by the T-lymphocytes.

8. The method of claim 5, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or gamma-Interferon mRNA synthesized by the T-lymphocytes.

9. The method of claim 6, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or gamma-Interferon mRNA synthesized by the T-lymphocytes.

10. The method of claim 1, further comprising calibrating the mass spectrometer based on candidate antigenic peptides predicted by an expression profile in a database.

11. The method of claim 1, further comprising detecting a T cell in the individual.

12. The method of claim 11, wherein the detecting comprises labelling the T cell with a complex comprising the antigenic peptides selected in step e), the HLA-subtypes, and a streptavidin-conjugated with a probe.

13. The method of claim 12, wherein the complex is a tetramer.

14. The method of claim 13, wherein the tetramer is prepared by biotinylating the HLA-subtypes and mixing the biotinylated HLA-subtypes bound with the antigenic peptides selected in step e) with the conjugated streptavidin.

15. The method of claim 14, wherein the conjugated streptavidin is streptavidin-phycoerythrin (PE) or streptavidin-allophycocyanin (APC).

16. The method of claim 15, wherein the detecting further comprises staining a peripheral blood mononuclear cell (PBMC) from the individual using the tetramer.

17. The method of claim 11, wherein the T cell is a CD8.sup.+ T cell.

18. The method of claim 1, further comprising formulating the synthesized antigenic peptides for administering to the individual.

19. The method of claim 1, further comprising isolating the one or more antigenic peptides identified in step d).

20. The method of claim 19, wherein the isolating comprises binding the one or more antigenic peptides bound to the HLA-subtypes with an anti-HLA-subtypes antibody.

21. The method of claim 20, wherein the anti-HLA-subtypes antibody is a monoclonal antibody W6/32 specific for HLA class I or a monoclonal antibody BB7.2 specific for HLA-A2.

22. The method of claim 21, wherein the isolating further comprising separating the one or more antigenic peptides by reversed phase HPLC.

23. The method of claim 1, wherein the tumor sample and the corresponding healthy tissue sample are embedded frozen tissues.

24. The method of claim 1, wherein the one or more genes identified by the microarray analysis is verified by the RT-PCR.

25. The method of claim 1, wherein the HLA-subtypes comprises HLA-A*02, HLA-A*68, HLA-B*18, and HLA-B*44.

26. The method for identifying antigenic peptides for use to induce a CTL immune response in an individual of claim 1, consisting of: a) determining HLA-subtypes of the individual, b) obtaining a tumor sample and a corresponding healthy tissue sample from the individual, c) identifying one or more genes that are overexpressed in the tumor sample relative to the healthy tissue sample using microarray analysis or RT-PCR, d) identifying one or more antigenic peptides bound to the HLA-subtypes in the tumor sample by mass spectrometry, e) selecting only the antigenic peptides from step d), which are encoded by an overexpressed gene from step c), wherein the antigenic peptides are selected from the group consisting of ALLNIKVKL (SEQ ID NO: 1), YVDPVITSI (SEQ ID NO: 3), SVASTITGV (SEQ ID NO:4), MTSALPIIQK (SEQ ID NO: 5), MAGDIYSVFR (SEQ ID NO: 6), ALAAVVTEV (SEQ ID NO: 7), and GIGSRGDRS (SEQ ID NO: 8), and f) synthesizing said antigenic peptides selected in step e), wherein the CTL immune response is induced in the individual.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention are displayed and explained in the figures and the example below.

(2) FIG. 1 shows the expression analysis of selected genes by means of quantitative RT-PCR;

(3) FIG. 2 shows the detection of keratin 18-specific CD8+ T-lymphocytes.

MODES OF CARRYING OUT THE INVENTION

Example 1

(4) Patient Samples

(5) Samples of patients having histologically confirmed renal cell carcinoma were obtained from the department of urology, University of Tubingen. Both patients had not received preoperative therapy. Patient No. 1 (in the following designated RCC01) had the following HLA-typing: HLA-A*02 A68 B*18 B*44; patient No 2 (in the following designated RCCI3) HLA-A*02 A*24 B*07 B*40.

(6) Isolation of MHC Class 1-Bound Peptides

(7) Shock-frozen tumor samples were processed as described in Schirle, M. et al., Identification of tumor-associated MHC-class I ligands by a novel T-cell-independent approach, 2000, European Journal of Immunology, 30: 2216-2225. Peptides were isolated according to standard protocols using monoclonal antibody W6/32 being specific for HLA class I or monoclonal antibody BB7.2 being specific for HLA-A2. Production and utilization of these antibodies is described by Barnstable, C. J. et al., Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens—New tools for genetic analysis, 1978, Cell, 14:9-20 and Parham, P. & Brodsky, F. M., Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLAA28, 1981, Hum. Immunol., 3: 277-299.

(8) Mass Spectrometry

(9) Peptides from tumor tissue of patient RCCO1 were separated by reversed phase HPLC (SMART-system, μRPC C2/C18 SC 2.1/19, Amersham Pharmacia Biotech) and fractions were analyzed by nanoESI MS. In doing so it was proceeded as described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T-cell-independent approach, 2000, European Journal of Immunology, 30: 2216-2225.

(10) Peptides from tumor tissue of patient RCC13 were identified by online capillary LCMS as mentioned above with minor modifications: Sample volumes of about 100 μl were loaded, desalted and preconcentrated on a 300 pm*5 mm C18 μ-precolumn (LC packings). A syringe pump (PHD 2000, Harvard Apparatus, Inc.) equipped with a gastight 100 pμl-syringe (1710 RNR, Hamilton), delivered solvent and sample at 2 μl/min. For peptide separation, the preconcentration column was switched in line with a 75*250 mm C-18-column (LC packings). Subsequently 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 using a TEE-piece (ZT1C, Valco) and a 300 μm*150 mm C-18-column.

(11) A blank run was always included to ensure that the system was free of residual peptides. On-line fragmentation was performed as described and fragment spectra were analyzed manually.

(12) Database searches (NCBInr, EST) were made using MASCOT.

(13) Preparation of RNA

(14) Fragments of normal and malignant renal tissue were dissected, shock-frozen, ground by a mortar and pestle under liquid nitrogen and homogenized with a rotary homogenizer (Heidolph instruments) in TRIZOL (Life Technologies). Total RNA was prepared according to the manufacturer's protocol followed by a clean-up with RNeasy (QIAGEN). Total RNA from human tissues were obtained commercially (Human total RNA Master Panel II, Clontech).

(15) High-Density Oligonucleotide Micro-Array Analysis

(16) Double-stranded DNA was synthesized from 40 μg of total RNA using superscript RT II reverse transcriptase (Life Technologies). The primer (Eurogentec) were given by the Affymetrix manual. In vitro transcription was performed using the BioArray™ High Yield™ RNA Transcript Labeling Kit (ENZO Diagnostics, Inc.); subsequently, fragmentation and hybridization were carried out on Affymetrix HuGeneFL gene chips, and staining with a streptavidin-phycoerythrin and biotinylated anti-streptavidin-antibody followed the manufacturer's protocols (Affymetrix). The Affymetrix GeneArray Scanner was used and data were analyzed with the Microarray Analysis Suite 4.0 Software.

(17) Real Time RT-PCR

(18) cDNA generated for microarray analysis was used for quantitative PCR analysis.

(19) Each gene was run in duplicates (40 cycles, 95° C.×15 s, 60° C.×1 min) using SYBRGreen chemistry on the ABI PRISM 7700 sequence detection system (Applied Biosystems). Samples were independently analyzed two to three times. Primers (MWGBiotech) were selected to flank an Intron and PCR efficiencies were tested for all primer pairs and found to be close to 1.

(20) 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). Data analysis involved the delta CT method for relative quantification.

Laser Capture Microdissection

(21) Embedded frozen tissue specimens were cut at 6 μm thickness and transferred in 70% ethanol for about 15 min. Slides were incubated 90 seconds in Mayer's hematoxylin (Merck), rinsed in water, incubated for 1 min in 70% ethanol, 1 min in 95% ethanol, 30 seconds in 1% alcoholic eosin Y (Sigma), 2×2 min in 95% ethanol, 2×2 min in 100% ethanol and finally 2×2 min in xylene. After air drying for 15 minutes, slides were stored under dry conditions. Normal malignant epithelial tubular cells and carcinoma cells were isolated by a Laser Capture Microdissection (LCM) using the PixCell II LCM system (Arcturus Engineering). Total RNA was extracted in 400 μl TRIZOL.

(22) PBMC, Tetramer Production and Flow Cytometry

(23) Peripheral blood mononuclear cells (in the following PBMC) from two healthy donors (HDI and HD2), which were serologically typed as CMV-positive, were isolated by gradient centrifugation (FicoLite H) and frozen.

(24) HLA-A *0201 tetrameric complexes were produced as described by Altman et al., 1996, Phenotypic analysis of antigen-specific T-lymphocytes, Science 274: 94-96, as follows: The HLA-A2-binding peptides used for the refolding were ALLNIKVKL (SEQ ID NO: 1) from keratin 18 and NLVPMVATV (SEQ ID NO: 2) from pp65 HCMVA. Tetramers were assembled by mixing biotinylated monomers with streptavidin-PE or streptavidin-APC and 2-3×10.sup.6 cells were incubated 30 min at 4° C. with both tetramers: 10 μg/ml for each monomer in PBS, 0.01% NaN3, 2 mM EDTA, 50% fetal calf serum). Then, monoclonal antibodies Anti-CD4-FITC (Coulter-Immunotech) and Anti-CD8-PerCP (Becton Dickinson) were added for 20 min. After three washes, samples were fixed in FACS buffer, 1% formaldehyde. Four-color analysis was performed on a FACScalibur cytometer (Becton Dickinson).

(25) Results

(26) The expression of approximately 7,000 genes in tumors and corresponding normal tissue of two renal cell carcinoma was analyzed. Between 400 and 500 genes were found to be overexpressed or selectively expressed in the tumor. 70 genes were overexpressed in the tumors of both patients. In patient I, 268 overexpressed and 129 exclusively expressed genes were found. Most of the overexpressed genes are cancer-related, i.e., either oncogenes, tumor suppressor genes or genes already described as overexpressed in cancer, such as CCND1, CA9, cerebrosidesulfotransferase and parathyroid hormone-like hormone. The cancer-associated adipose differentiation-related protein (ADFP) or adipophilin, showed the second highest degree of overexpression. In addition, this protein was shown to be highly overexpressed in tumorous tissue in comparison to normal tissue of other organs, that is not only in comparison to normal renal tissue.

(27) To verify data obtained by microarray analysis the expression of selected genes was analyzed by quantitative PCR. In FIG. 1, the expression-analysis of selected genes by means of RT-PCR is shown. The RT-PCR was performed using the same cDNA as generated for microarray analysis. Copy numbers are relative to 18S rRNA and normalized to the normal tissue (=1) of each patient. The black bars correspond to the numbers in tumor tissue of patient RCC01, the white bars represent the numbers of normal tissues and the angular-striped gray bars the numbers in tumor tissue of patient RCC13.

(28) It was shown that overexpression of adipophilin (ADFP) and cycline D1 (CCND1) as proven by microarray could be confirmed by quantitative PCR. Further, it was demonstrated that ets-1 (ETS1) was expressed equally both in normal and in tumor tissue. Further, relative expression levels detected by both techniques were roughly comparable.

(29) For example, adipophilin was overexpressed in tumor tissue of patient RCCOI by a factor of 29.1 as proven by means of microarray, compared to 18.1 as measured by means of quantitative PCR (see FIG. 1). With patient RCC13, by means of microarray analysis a factor of 11.4 and by means of quantitative PCR a factor of 6.7 could be demonstrated (see FIG. 1). Galectin 2 (LGALS2) was overexpressed in patient RCCO1 and keratin-18 (KRT18) in patient RCC13. An exception to the congruence between microarray and quantitative PCR was the overexpression of KIAA0367 and met proto-oncogene (MET) in patient RCC13.

(30) Identification of MHC Class 1-Ligands

(31) A total of 85 ligands could be obtained from tumor tissue, which were bound to HLA-subtypes HLA-A*02, HLA-A*68, HLA-B*18 or HLA-B*44. Peptides that bind to HLAA*02 reflected the allele-specific peptide motif (Leucine, Valine, Isoleucine, Alanine, Methionine on position 2, Leucine, Valine, Isoleucine or Alanine at the C-Terminus). Most ligands were derived from abundantly expressed housekeeping proteins, but ligands from proteins with reported tumor association could be detected also, for example, YVDPVITSI (SEQ ID NO: 3) derived from met proto-oncogene, ALLNIKVKL (SEQ ID NO: 1) derived from keratin 18, and SVASTITGV (SEQ ID NO: 4) from adipophilin.

(32) HLA-A*68 ligands were identified by their anchor amino acids Threonine, Isoleucine, Valine, Alanine or Leucine on position 2 and arginine or lysine at the C-terminus. Two other ligands from adipophilin were found among HLA-A*68-presented peptides: MTSALPIIQK (SEQ IDS NO: 5) and MAGDIYSVFR (SEQ ID NO: 6). Ligands carrying glutamic acid on position 2 were assigned to HLA-B*44; since the peptide motif of HLA-B*18 is unknown, a distinction between ligands of these two HLA-B-molecules was not possible.

(33) Comparison of microarray data with the isolated ligands indicated 10 overexpressed genes as sources for MHC-ligands: adipophilin, KIAA0367, SEC14-like 1, B-cell translocation gene 1, aldolase A, cycline D1, annexin A4, catenin alpha 1, galectin 2 and LMP2. Three of them were also included in the SEREX database: KIAA0367, aldolase A and catenin alpha 1.

(34) A most interesting ligand could be identified from patient RCC13 (ALAAVVTEV SEQ ID NO: 7)) encoded by a “Reading frame” shifted by one nucleotide compared to DEAD/H-box polypeptide 3 (DDX3). ALAAVVTEV (SEQ IF NO: 7) is encoded by the nucleotides 317 to 343 of the coding strand of DDX3, whereas nucleotides 316 to 342 are coding for GIGSRGDRS (SEQ ID NO: 8) of the DDX3 protein.

(35) Detection of Specific T-ceHs in Normal CD8.sup.+ T-Cell Repertoire

(36) PBMC from 6 HLA-A2 positive renal cell carcinoma patients were tested for reaction against four of the relevant peptides: HLA-A*02-restricted ligands from adipophilin, keratin 18, K1AA0367 and met-proto-oncogene. In doing so, a very sensitive quantitative PCT assay was carried out to detect γ-Interferon-mRNA production by CD8.sup.+ T-cells following a 7 day-in vitro-sensitization with peptide. Sporadic responses were seen after stimulation with met-proto-oncogene or keratin 18 or adipophilin peptides.

(37) Staining of PBMC of tumor patients and healthy individuals with HLA-A*0201 tetramers was performed with tetramers reconstituted either with adipophilin, keratin 18 or met-proto-oncogene.

(38) FIG. 2 shows the detection of keratin 18-specific T-lymphocytes. For this purpose, PBMC from four healthy HLA-A*02.sup.+ donors (HD 1, 2, 4, 6) were simultaneously stained with HLA-A2/keratin 18-PE tetramers, HLA-A2/CMV-APC tetramers, CD8-PerCP and CD4-FITC. Dot plots show samples from one of three independent experiments for 1×10.sup.6 PBMC. In the plots, percentage of tetramer.sup.+-cells within the CD8.sup.+ CD4.sup.− population are indicated.

(39) Unexpectedly, a significant population of CD8.sup.+ T-lymphocytes specific for keratin 18 (between 0.02% and 0.2% of CD8.sup.+ T-cells) was found in 4 out of 22 healthy individuals. This population did not stain with a CMV tetramer showing that the binding of keratin 18 tetramer was specific.

(40) To summarize, it can be concluded that CD8.sup.+ T-lymphocytes specific for the keratin 18-peptide are contained in the human T-cell repertoire.