PRAME derived peptides and immunogenic compositions comprising these
10450356 ยท 2019-10-22
Assignee
Inventors
- Jan Kessler (Leiden, NL)
- Marieke Griffioen (Alphen a/d Rijn, NL)
- Cornelis Johannes Maria Melief (Haarlem, NL)
- Jan Wouter Drijfhout (Leiden, NL)
Cpc classification
A61K39/4611
HUMAN NECESSITIES
C07K14/4748
CHEMISTRY; METALLURGY
A61K2039/57
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The invention relates to a peptide having a length of no more than 100 amino acids and comprising at least 19 contiguous amino acids from the amino acid sequence of the human PRAME protein, wherein the peptide comprises at least one HLA class II epitope and at least one HLA class I epitope from the amino acid sequence of the human PRAME protein and to its use as such or in a composition as a medicament for the treatment and/or prevention of cancer.
Claims
1. An immunogenic pharmaceutical composition comprising (a) a peptide having a length of 27 to 35 amino acids and comprising at least 27 contiguous amino acids from the amino acid sequence of the human PRAME protein and (b) an immune-stimulating amount of a pharmaceutically acceptable adjuvant, wherein the peptide comprises at least one HLA class II epitope and at least one HLA class I epitope from the amino acid sequence of the human PRAME protein, wherein said HLA class I epitope is capable of activating a CD8+ CTL in a human cancer patient and/or in a healthy control and is represented by the amino acid sequence selected from the group consisting of: amino acid sequence including amino acids at positions 100-108 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 113-122 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 142-151 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 190-198 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 258-267 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 371-380 of SEQ ID NO: 21; and amino acid sequence including amino acids at positions 425-433 of SEQ ID NO: 21; wherein said HLA class II epitope is capable of activating a CD45RO positive CD4+ Th cell and is represented by the amino acid sequence selected from the group consisting of: amino acid sequence including amino acids at positions 98-124 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 116-142 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 133-159 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 181-207 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 194-220 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 234-255 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 247-273 of SEQ ID NO: 21; amino acid sequence including amino acids at positions 353-379 of SEQ ID NO: 21; and amino acid sequence including amino acids at positions 424-450 of SEQ ID NO: 21, and wherein the contiguous amino acid sequence from the human PRAME protein is selected from the group consisting of: amino acid sequences SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 16, and 18.
2. The immunogenic pharmaceutical composition according to claim 1, wherein the length of the contiguous amino acid sequence is 30-35 amino acids.
3. The immunogenic pharmaceutical composition according to claim 1, wherein the HLA class I epitope is an HLA-A2 epitope.
4. The immunogenic pharmaceutical composition according to claim 1, wherein the peptide consists of a contiguous amino acid sequence from the human PRAME protein that is selected from the group consisting of amino acid sequences SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 16, and 18.
5. The immunogenic pharmaceutical composition according to claim 1, wherein said peptide is modified by addition at the N- and/or C-terminus, of one amino acid.
6. The immunogenic pharmaceutical composition according to claim 1, comprising at least two different peptides as defined in claim 1.
7. The immunogenic pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable adjuvant acts via a Toll-like receptor.
8. The immunogenic pharmaceutical composition according to claim 1 for the treatment or prevention of cancer.
9. The immunogenic pharmaceutical composition according to claim 1, wherein the cancer is a cancer in which the human PRAME protein is overexpressed.
10. The immunogenic pharmaceutical composition according to claim 1, wherein the cancer is selected from the group consisting of: melanoma, lymphoma, papillomas, breast or cervical carcinomas, acute and chronic leukemias, medulloblastoma, non-small cell lung carcinoma, head and neck cancer, renal carcinoma, pancreatic carcinoma, prostate cancer, small cell lung cancer, multiple myeloma, sarcomas and hematological malignancies like chronic myeloid leukemia and acute myeloid leukemia.
11. The immunogenic pharmaceutical composition according to claim 9, wherein the cancer is selected from the group consisting of: melanoma, lymphoma, papillomas, breast or cervical carcinomas, acute and chronic leukemias, medulloblastoma, non-small cell lung carcinoma, head and neck cancer, renal carcinoma, pancreatic carcinoma, prostate cancer, small cell lung cancer, multiple myeloma, sarcomas and hematological malignancies like chronic myeloid leukemia and acute myeloid leukemia.
12. The immunogenic pharmaceutical composition according to claim 1, wherein the length of the contiguous amino acid sequence is 33-35 amino acids.
13. The immunogenic pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable adjuvant is synthetic.
14. The immunogenic pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of: Gram positive bacterial glycolipids, fimbriae, outer membrane proteins, heatshock proteins, mycobacterial lipoarabinomannans, dsRNA, poly(I:C), Gram negative glycolipids, viral coat or envelope proteins, taxol or derivatives thereof, hyaluronan containing oligosaccharides or fibronectins, bacterial flagellae or flagellin, mycobacterial lipoproteins, group B Streptococcus heat labile soluble factor (GBS-F), Staphylococcus modulins, and imidazoquinolines.
15. The immunogenic pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of: dsRNA, poly(I:C), unmethylated CpG DNA, IC31, IMSAVAC, and Montanide.
16. The immunogenic pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable adjuvant is physically linked to the peptide.
17. The immunogenic pharmaceutical composition according to claim 1, further comprising at least one immune modulator.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
(5) In the current invention the different aspects that are required for the induction of an efficient and successful vaccine-induced T cell response against PRAME expressing cancer cells in patients are combined for the design and selection of optimal PRAME derived vaccine peptides. An optimal PRAME vaccine peptide should encompass at least one, but preferably more, HLA class I presented cytotoxic T lymphocyte (CTL) epitope(s) capable to induce a CTL response in patients, together with at least one PRAME-derived peptide with proven capacity to elicit a CD4.sup.+ Th lymphocyte response. The experimental section provides the parameters required for the optimal design and choice of PRAME derived peptides for vaccination in terms of sequence and length/size. The experimental section discloses both identification and confirmation of HLA class I presented CTL epitopes and CD4.sup.+ Th lymphocyte reactivity inducing peptides, in vitro and in vivo, that are present in the full length PRAME protein and which can be combined in peptides having an optimal length of 19-45 amino acids.
Example 1: Identification of HLA Class I Presented Peptides from PRAME
(6) Synthetic Production of Peptides
(7) All peptides used in these studies were synthesized by solid phase strategies on an automated multiple peptide synthesizer (Abimed AMS 422) using standard Fmoc chemistry. Short peptides for CTL inductions were dissolved in 20 ?l DMSO, diluted in 0.9% NaCl to a peptide concentration of 1 mg/ml and stored at ?20? C. before usage. The fluorescein-labeled reference peptides, used in the HLA class I peptide binding assays, were synthesized as Cys-derivative. Labeling was performed with 5-(iodoacetamido)fluorescein (Fluka Chemie AG, Buchs, Switzerland) at pH 7.5 (Na-phosphate in water/acetonitrile 1:1 v/v). The labelled peptides were desalted over Sephadex G-10 and further purified by C18 RP-HPLC. Labelled peptides were analysed by mass spectrometry. The 27-mer and 22-mer polypeptides used for in vitro proteasome digestion analysis and analysis of CD4.sup.+ Th lymphocyte reactivity were synthesized as described above, purified by reversed phase-HPLC in an acetonitrile-water gradient and lyophilized from acetonitrile-water overnight. Purity was confirmed by mass spectrometry.
(8) Pre-Selection of PRAME Peptides for HLA Class I Binding Measurements
(9) A selection of PRAME peptides with a length of 8, 9, 10 or 11 amino acids with potential binding capacity for the HLA class I molecules that are most predominant was made using the peptide binding prediction algorithms BIMAS (Parker, et al., 1994, J. Immunol. 152:163) and SYFPEITHI. These computer algorithms search for peptides contained in the full length PRAME protein complying to the binding motifs of the HLA class I molecule of interest. HLA class I molecules were chosen with high or at least moderate prevalences in the human population, being HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B35, HLA-B60, HLA-B61 and HLA-B62. Prevalences among the human populations of these HLA class I molecules are shown in Table 1 below.
(10) Using the algorithm, the full length PRAME protein was screened for peptides with a predicted (efficient) binding capacity for the chosen HLA class I molecules. The PRAME peptides (length 9, 10 or 11 aa.) with a high predicted binding capacity were synthetically produced to enable actual experimental determination of their binding capacity in competition-based HLA class I binding assays. Because a high prediction score for binding to a certain HLA class I molecule does not necessarily correlate with actual high affinity binding (as has been shown by Kessler et al., 2003, Hum Immunol. 64:245) such binding measurements are required for the assessment of the binding capacity.
(11) TABLE-US-00001 TABLE 1 Frequency distribution of HLA I antigens expressed as percentages among major populations.sup.a HLA Population class I Black Caucasoid Oriental Amerindian A1 9 26 7 11 A2 29 44 47 43 A3 13 22 6 8 A11 3 13 30 4 A24 6 20 42 52 A68 18 8 3 12 B7 15 17 7 5 B8 9 14 3 2 B14 7 6 1 3 B35 11 20 10 32 B60 1 6 17 5 B61 0 6 9 23 B62 2 8 16 21 .sup.aPhenotype frequencies for the HLA antigens have been deduced using the gene frequencies as given by: Marsh et al., The HLA FactsBook., 1999.
Determination of HLA Class I Peptide Binding Capacity
(12) For the experimental measurement of HLA class I binding capacity, HLA class I competition-based cellular binding assays were used that have been developed for HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B35, HLA-B60, HLA-B61 and HLA-B62 (Kessler et al., 2003, Hum Immunol. 64:245). EBV transformed human B cells (B-LCL) were used that were stripped from their naturally presented HLA class I peptides by mild acid treatment. B-LCL were harvested and washed in phosphate buffered saline (PBS) and the pellet (2-15?10.sup.6 cells) was put on ice for 5 min. The elution was performed by incubating the cells for exactly 90 s in ice-cold citric-acid buffer (1:1 mixture of 0.263 M citric acid and 0.123 M Na.sub.2HPO.sub.4, adjusted to the pH listed in Table 2). Immediately thereafter, cells were buffered with ice-cold IMDM containing 2% FCS, washed once more in the same medium and resuspended at a concentration of 4?10.sup.5 cells/ml in IMDM medium containing 2% FCS and 2 ?g/ml human ?.sub.2-microglublin (?.sub.2M) (Sigma, St. Louis, Mo., USA).
(13) Eight serial twofold dilutions of each competitor test peptide in PBS/BSA 0.5% were made (highest concentration 600 ?M, 6-fold assay concentration). In the assay, test peptides were tested from 100 ?M to 0.8 ?M. Fluoresceine (Fl)-labeled reference peptides that are used in the different HLA class I competition assays and their source are listed in Table 2. These peptides, which have established high binding affinity in the HLA class I molecule under study, were dissolved in PBS/BSA 0.5% at 6-fold final assay concentration. In a well of a 96-well V-bottom plate 25 ?l of competitor (test) peptide was mixed with 25 ?l Fl-labeled reference peptide. Subsequently, the stripped B-LCL were added at 4?10.sup.4/well in 100 ?l/well. After incubation for 24 h at 4? C., cells were washed three times in PBS containing 1% BSA, fixed with 0.5% paraformaldehyde, and analyzed with FACScan flowcytometry (Becton Dickinson) to measure the mean fluorescence (MF). The percentage inhibition of Fl-labeled reference peptide binding was calculated using the following formula:
(1?(MF.sub.reference+competitor peptide?MF.sub.background)/(MF.sub.reference peptide?MF.sub.background))?100%.
The binding affinity of competitor peptide is expressed as the concentration that inhibits 50% binding of the Fl-labeled reference peptide (IC.sub.50). IC.sub.50 was calculated applying non-linear regression analysis. An IC.sub.50 5 ?M was considered high affinity binding, 5 ?M<IC.sub.50?about 15 ?M was considered intermediate affinity binding, about 15 ?M<IC.sub.50?100 ?M was judged low affinity binding and IC.sub.50>100 ?M was regarded as no binding.
(14) TABLE-US-00002 TABLE2 CharacteristicsofthedifferentHLAclassIbindingassays. Referencepeptidesusedintheassays FL-labeledseq. Originalseq. (SEQIDNOS (SEQIDNOS 23-34, 35-46 respectively, respectively, HLAclassI inorderof inorderof B-LCLcelllineusedintheassay Allele appearance) [pep.] appearance) Name HLAclassItype A1(A*0101) YLEPAC(FI)AKY 150nM YLEPAIAKY CAA A*0101,B*0801,Cw*0701 A2(A*0201) FLPSDC(FI)FPSV 150nM FLPSDFFPSV JY A*0201,B*0702,Cw*0702 A3(A*0301) KVFPC(FI)ALINK 150nM KVFPYALINK EKR A*0301,B*0702,Cw*0702 A11(A*1101) KVFPC(FI)ALINK 150nM KVFPYALINK BVR A*1101,B*3501,Cw*0401 A24(A*2402) RYLKC(FI)QQLL 150nM RYLKDQQLL Vijf A*2402;B*0702,Cw*0702 A68(A*6801) KTGGPIC(FI)KR 150nM KTGGPIYKR A68HI A*6801,B*4402,Cw*0704 B7(B*0702) APAPAPC(FI)WPL 150nM APAPAPSWPL JY A*0201,B*0702,Cw*0702 B8(B*0801) FLRGRAC(FI)GL 50nM FLRGRAYGL Vavy A*0101,B*0801,Cw*0701 B35(B*3501) NPDIVC(FI)YQY 150nM NPDIVIYQY BVR A*1101,B*3501,Cw*0401 B60(B*4001) KESTC(FI)HLVL 125nM KESTLHLVL DKB A*2402,B*4001,Cw*0304 B61(B*4002) GEFGGC(FI)GSV 50nM GEFGGFGSV Swei007 A*2902,B*4002,Cw*0202 B62(B*1501) YLGEFSC(FI)TY 150nM YLGEFSITY BSM A*0201,B*1501,Cw*0304
Results of the HLA Class I Binding Assays
(15) The actual binding measurements revealed that 49 PRAME peptides (9 or 10 aa. long) displayed an high or intermediate affinity for HLA-A2 (Table 3a) and, as shown in Table 3b, 93 peptides (8-, 9-, 10-, 11-mers) had a high or intermediate binding capacity for the other HLA class I molecules (HLA-A1, HLA-A3, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B35, HLA-B60, HLA-B61 and HLA-B62). These peptides with a proven HLA class I binding capacity were further analysed for their enzymatic liberation from their flanking protein sequence by proteasomal cleavage using the results of the proteasome digestion analysis (
(16) TABLE-US-00003 TABLE3A HighandintermediatebindingHLA-A2(*0201) peptidesfromPRAME. Binding Start.sup.a Sequence.sup.b Length.sup.c (IC.sub.50.sup.d) 25 RLVELAGQSL 10 11.1 33 SLLKDEALAI 10 14.0 34 LLKDEALAI 9 10.2 39 ALAIAALEL 9 5.1 39 ALAIAALELL 10 9.0 47 LLPRELFPPL 10 2.1 71 AMVQAWPFTC 10 10.4 91 HLHLETFKA 9 11.1 99 AVLDGLDVL 9 13.4 99 AVLDGLDVLL 10 9.4 100 VLDGLDVLL 9 5.2 100 VLDGLDVLLA 10 11.9 103 GLDVLLAQEV 10 15.2 142 SLYSFPEPEA 10 1.9 182 FLKEGACDEL 10 3.0 186 GACDELFSYL 10 10.6 190 ELFSYLIEKV 10 4.5 214 KIFAMPMQDI 10 7.2 242 CTWKLPTLA 9 9.3 248 TLAKFSPYL 9 4.6 258 QMINLRRLLL 10 4.0 284 YIAQFTSQFL 10 10.4 292 FLSLQCLQAL 10 2.5 294 SLQCLQALYV 10 3.2 300 ALYVDSLFF 9 2.7 300 ALYVDSLFFL 10 1.7 301 LYVDSLFFL 9 6.3 308 FLRGRLDQLL 10 9.6 320 VMNPLETLSI 10 8.6 326 TLSITNCRL 9 13.2 333 RLSEGDVMHL 10 6.1 350 QLSVLSLSGV 10 13.3 355 SLSGVMLTDV 10 9.9 360 MLTDVSPEPL 10 5.6 371 ALLERASATL 10 12.9 390 ITDDQLLAL 9 9.2 394 QLLALLPSL 9 2.9 410 TLSFYGNSI 9 11.0 419 SISALQSLL 9 5.7 422 ALQSLLQHL 9 14.2 422 ALQSLLQHLI 10 3.2 425 SLLQHLIGL 9 3.7 432 GLSNLTHVL 9 6.8 435 NLTHVLYPV 9 2.5 454 TLHLERLAYL 10 12.2 462 YLHARLRELL 10 13.3 462 YLHARLREL 9 6.2 466 RLRELLCEL 9 14.0 470 LLCELGRPSM 10 10.5 .sup.aPostion in PRAME of the N-terminal amino acid (aa.) of the peptide. Peptides are sorted by their starting aa. .sup.bAa. sequence of the peptide .sup.cLength of the peptide .sup.dIC.sub.50 is peptide concentration needed to inhibit binding of FL-labeled reference peptide for 50% (IC.sub.50 in ?M). Peptides with IC.sub.50 ? about 15 ?M are considered to be potential CTL epitopes with respect to their binding affinity.
(17) TABLE-US-00004 TABLE3B HighandintermediateaffinitybindingHLAclass I(nonHLA-A2)peptidesfromPRAME. HLA Binding Start.sup.a Sequence.sup.b Length.sup.c ClassI.sup.d (IC.sub.50.sup.e) 136 WSGNRASLY 9 HLA-A1 4.3 165 STEAEQPFI 9 HLA-A1 1.4 247 PTLAKFSPY 9 HLA-A1 8.5 267 LSHIHASSY 9 HLA-A1 1.0 275 YISPEKEEQY 10 HLA-A1 3.0 292 FLSLQCLQALY 11 HLA-A1 1.0 293 LSLQCLQALY 10 HLA-A1 2.9 294 SLQCLQALY 9 HLA-A1 2.0 302 YVDSLFFLR 9 HLA-A1 1.4 334 LSEGDVMHL 9 HLA-A1 6.3 361 LTDVSPEPLQ 10 HLA-A1 3.8 361 LTDVSPEPLQA 11 HLA-A1 3.5 390 ITDDQLLAL 9 HLA-A1 1.0 390 ITDDQLLALL 10 HLA-A1 1.5 405 CSQLTTLSFY 10 HLA-A1 >1 433 LSNITHVLY 9 HLA-A1 <1 439 VLYPVPLESY 10 HLA-A1 10.9 453 GTLHLERLAY 10 HLA-A1 2.0 454 TLHLERLAY 9 HLA-A1 10.1 5 RLWGSIQSRY 10 HLA-A3 1.59 5 RLWGSIQSR 9 HLA-A3 1.13 16 SMSVWTSPR 9 HLA-A3 <1 28 ELAGQSLLK 9 HLA-A3 3.14 41 AIAALELLPR 10 HLA-A3 10.75 80 CLPLGVLMK 9 HLA-A3 <1 107 LLAQEVRPRR 10 HLA-A3 14.0 118 KLQVLDLRK 9 HLA-A3 2.15 190 ELFSYLIEK 9 HLA-A3 1.42 194 YLIEKVKRK 9 HLA-A3 3.49 194 YLIEKVKRKK 10 HLA-A3 14.00 198 KVKRKKNVLR 10 HLA-A3 7.50 204 NVLRLCCKK 9 HLA-A3 13.50 205 VLRLCCKKLK 10 HLA-A3 1.30 242 CTWKLPTLAK 10 HLA-A3 <1 255 YLGQMINLRR 10 HLA-A3 4.50 261 NLRRLLLSH 9 HLA-A3 3.50 300 ALYVDSLFF 9 HLA-A3 8 333 RLSEGDVMH 9 HLA-A3 16.00 429 HLIGLSNLTH 10 HLA-A3 4.00 432 GLSNLTHVLY 10 HLA-A3 4.07 439 VLYPVPLESY 10 HLA-A3 2.67 459 RLAYLHARLR 10 HLA-A3 1.00 13 RYISMSVWTS 10 HLA-A24 5.8 52 LFPPLFMAAF 10 HLA-A24 <1 60 AFDGRHSQTL 10 HLA-A24 5.5 77 PFTCLPLGVL 10 HLA-A24 2.1 85 VLMKGQHLHL 10 HLA-A24 15 96 TFKAVLDGL 9 HLA-A24 8.6 173 IPVEVLVDLF 10 HLA-A24 <1 215 IFAMPMQDI 9 HLA-A24 1.8 251 KFSPYLGQMI 10 HLA-A24 2.5 254 PYLGQMINL 9 HLA-A24 <1 283 QYIAQFTSQF 10 HLA-A24 8.2 287 QFTSQFLSL 9 HLA-A24 1.0 301 LYVDSLFFL 9 HLA-A24 <1 307 FFLRGRLDQL 10 HLA-A24 1.8 412 SFYGNSISI 9 HLA-A24 <1 447 SYEDIHGTL 9 HLA-A24 <1 459 RLAYLHARL 9 HLA-A24 <1 461 AYLHARLREL 10 HLA-A24 <1 466 RLRELLCEL 9 HLA-A24 <1 494 TFYDPEPIL 9 HLA-A24 <1 150 EAAQPMTKK 9 HLA-A*6801 pred. 150 EAAQPMTKKR 10 HLA-A*6801 pred. 302 YVDSLFFLR 9 HLA-A*6801 <1 113 RPRRWKLQVL 10 HLA-B7 <1 113 RPRRWKLQVL 10 HLA-B8 <1 258 QMINLRRLLL 10 HLA-B8 1.67 259 MINLRRLL 8 HLA-B8 <1 260 INLRRLLL 8 HLA-B8 <1 462 YLHARLREL 9 HLA-B8 <1 48 LPRELFPPL 9 HLA-B*3501 <1 48 LPRELFPPLF 10 HLA-B*3501 1.58 53 FPPLFMAAF 9 HLA-B*3501 <1 170 QPFIPVEVL 9 HLA-B*3501 2.83 173 IPVEVLVDL 9 HLA-B*3501 2.24 173 IPVEVLVDLF 10 HLA-B*3501 <1 186 GACDELFSY 9 HLA-B*3501 2.60 246 LPTLAKFSPY 10 HLA-B*3501 <1 253 SPYLGQMINL 10 HLA-B*3501 1.98 487 CPHCGDRTFY 10 HLA-B*3501 1.5 499 EPILCPCFM 9 HLA-B*3501 <1 36 KDEALAIAAL 10 HLA-B60 2.91 37 DEALAIAAL 9 HLA-B60 1.55 50 RELFPPLFM 9 HLA-B60 1.48 448 YEDIHGTLHL 10 HLA-B60 <1 37 DEALAIAAL 9 HLA-B61 <1 50 RELFPPLFM 9 HLA-B61 <1 50 RELFPPLFMA 10 HLA-B61 <1 94 LETFKAVL 8 HLA-B61 <1 89 GQHLHLETF 9 HLA-B62 2.39 300 ALYVDSLFF 9 HLA-B62 <1 316 LLRHVMNPL 9 HLA-B62 2.56 427 LQHLIGLSNL 10 HLA-B62 2.41 439 VLYPVPLESY 10 HLA-B62 1.66 .sup.aPostion in PRAME of the N-term; peptides are sorted by HLA molecule and start position. .sup.bAmino acid (aa.) sequence of the peptide .sup.cLength of the peptide .sup.dHLA class I molecule in which the peptide binds .sup.eIC.sub.50 peptide concentration that inhibits binding of FL-labeled reference peptide for 50% (IC.sub.50 in ?M). Peptides with IC.sub.50 < about 15 ?M are potential CTL epitopes, with respect to their binding affinity. Pred., indicates high binding affinity predicted, but not tested.
Example 2: Determination of Proteasomal Cleavage Sites in Full Length PRAME
(18) Materials and Methods In Vitro Proteasome Mediated Cleavage Analysis
(19) 20S proteasomes were purified from a B-LCL cell line as described by Groettrup et al. (J. Biol. Chem. 270:23808-23815; 1995). This cell type is known to contain immunoproteasomes. High LMP2 and 7 content was confirmed by 2-D immuno-blotting. To assess kinetics, digestions were performed with different incubation periods. Peptides (27 mers, 20 ?g) were incubated with 1 ?g of purified proteasome at 37? C. for 1 h, 4 h and 24 h in 300 ?l proteasome digestion buffer as described (Eggers, et al. 1995. J. Exp. Med. 182:1865). Trifluoroacetic acid was added to stop the digestion and samples were stored at ?20? C. before mass spectrometric analysis.
(20) Electrospray ionization mass spectrometry was performed on a hybrid quadrupole time-of-flight mass spectrometer, a Q-TOF (Micromass), equipped with an on-line nanoelectrospray interface with an approximate flow rate of 250 nL/min. Injections were done with a dedicated micro/nano HPLC autosampler, the FAMOS (LC Packings). Digestion solutions were diluted five times in water-methanol-acetic acid (95:5:1, v/v/v), and trapped on the precolumn (MCA-300-05-C8; LC Packings) in water-methanol-acetic acid (95:5:1, v/v/v). Washing of the precolumn was done for 3 min to remove the buffers present in the digests. Subsequently, the trapped analytes were eluted with a steep gradient going from 70% B to 90% B in 10 min, with a flow of 250 nl/min (A: water-methanol-acetic acid (95:5:1, v/v/v); B: water-methanol-acetic acid (10:90:1, v/v/v)). This low elution rate allows for a few additional MS/MS experiments if necessary during the same elution. Mass spectra were recorded from mass 50-2000 Da every second. The resolution allows direct determination of the monoisotopic mass, also from multiple charged ions. The peaks in the mass spectrum were searched in the digested precursor peptide using the Biolynx/proteins software (Micromass). The intensity of the peaks in the mass spectra was used to establish the relative amounts of peptides generated by proteasome digestion.
(21) Results of In Vitro Proteasome Mediated Cleavage Analysis
(22) Twentynine overlapping PRAME peptides (mostly 27-mers) that cover almost the entire PRAME aa. sequence, were digested in vitro with purified 20S proteasomes. Digestion intervals were 1 hr, 4 hr and 24 hr. Mass spectrometrical analysis of the digestion fragments revealed abundant and low abundant proteasomal cleavage sites within the digested PRAME peptides.
(23)
(24) The identification of the peptide fragments generated by in vitro proteasomal cleavage was used to assess the C-terminal generation of the high and intermediate affinity binding HLA class I peptides (Table 3a, 3b) on the one hand and the presence of the epitope as an intact fragment after proteasomal cleavage on the other hand.
(25) TABLE-US-00005 TABLE4 HLAclassIbindingpeptidesfromPRAMEthatarepresentasintact fragmentwiththecorrectC-terminusafterproteasomalcleavage. aa. HLA C-term. Intactin Start.sup.a End sequence.sup.b ClassI.sup.c generation.sup.d fragment.sup.e 16 24 SMSVWTSPR HLA-A3 seeEx.3(Note.sup.f) NT 33 42 SLLKDEALAI HLA-A2 ++ + 34 42 LLKDEALAI HLA-A2 ++ + 36 45 KDEALAIAAL HLA-B60 ++ ND 37 45 DEALAIAAL HLA-B60 ++ ND 37 45 DEALAIAAL HLA-B61 ++ ND 48 57 LPREIFPPLF HLA-B*3501 + ND 50 58 RELFPPLFM HLA-B60 ++ + 50 58 RELFPPLFM HLA-B61 ++ + 50 59 RELFPPLFMA HLA-B61 ++ + 52 61 LFPPLFMAAF HLA-A24 ++ ND 53 61 FPPLFMAAF HLA-B*3501 ++ ND 60 69 AFDGRHSQTL HLA-A24 + + 77 86 PFTCLPLGVL HLA-A24 ++ + 89 97 GQHLHLETF HLA-B62 ++ + 94 101 LETFKAVL HLA-B61 ++ + 99 108 AVLDGLDVLL HLA-A2 ++ + 100 108 VLDGLDVLL HLA-A2 ++ + 113 122 RPRRWKLQVL HLA-B7 + + 113 122 RPRRWKLQVL HLA-B8 + + 142 151 SLYSFPEPEA HLA-A2 ++ + 150 158 EAAQPMTKK HLA-A*6801 seeEx.3(Note.sup.f) NT 150 159 EAAQPMTKKR HLA-A*6801 seeEx.3(Note.sup.f) NT 170 178 QPFIPVEVL HLA-B*3501 ++ ND 190 198 ELFSYLIEK HLA-A3 seeEx.3(Note.sup.f) + 248 256 TLAKFSPYL HLA-A2 + + 254 262 PYLGQMINL HLA-A24 +/seeEx.3(Note.sup.f) + 253 262 SPYLGQMINL HLA-B*3501 +/seeEx.3(Note.sup.f) + 259 266 MINLRRLL HLA-B8 + + 258 267 QMINLRRLLL HLA-A2 + + 258 267 QMINLRRLLL HLA-B8 + + 260 267 INLRRLLL HLA-B8 + + 283 292 QYIAQFTSQF HLA-A24 ++ + 284 293 YIAQFTSQFL HLA-A2 ++ + 287 295 QFTSQFLSL HLA-A24 ++ ND 300 308 ALYVDSLFF HLA-A2 + + 300 308 ALYVDSLFF HLA-A3 + + 300 308 ALYVDSLFF HLA-B62 + + 300 309 ALYVDSLFFL HLA-A2 ++ + 301 309 LYVDSLFFL HLA-A2 ++ + 301 309 LYVDSLFFL HLA-A24 ++ + 326 334 TLSITNCRL HLA-A2 ++ + 334 342 LSEGDVMHL HLA-A1 + + 333 342 RLSEGDVMHL HLA-A2 + + 361 370 LTDVSPEPLQ HLA-A1 + + 361 371 LTDVSPEPLQA HLA-A1 + + 371 380 ALLERASATL HLA-A2 ++ + 390 399 ITDDQLLALL HLA-A1 + + 410 418 TLSFYGNSI HLA-A2 ++ + 412 420 SFYGNSISI HLA-A24 ++ + 425 433 SLLQHLIGL HLA-A2 ++ + 427 436 LQHLIGLSNL HLA-B62 + + 429 438 HLIGLSNLTH HLA-A3 + + 439 448 VLYPVPLESY HLA-A1 + + 439 448 VLYPVPLESY HLA-A3 + + 439 448 VLYPVPLESY HLA-B62 + + 459 467 RLAYLHARL HLA-A24 ++ + 462 470 YLHARLREL HLA-A2 + + 461 470 AYLHARLREL HLA-A24 + + 462 470 YLHARLREL HLA-B8 + + 462 471 YLHARLRELL HLA-A2 + + .sup.aPosition in PRAME of the N-terminus of the presented epitope. Peptides are sorted by start aa. .sup.baa. sequence of the peptide. .sup.cHLA class I molecule in which the peptide binds. .sup.dGeneration of C-terminus of the epitope after 1 h digestion: classification: abundant (++) present for >5%, low abundant (+) present for <5%. .sup.eIntact epitope found in digestion fragments after 1 h digestion: (+), present; (-), not present; (ND), could not be determined due to artificial ends of the synthetic input peptides; (NT), Not tested, but predicted to be abundantly made by Nardilysin. .sup.fThe C-terminus of PRA(190-198) is generated by a non-proteasomal cleavage pathway, involving first Nardilysin and subsequently Thimet oligopeptidase (TOP) as explained in Example 3 and FIG. 2. The C-termini of PRA(16-24), PRA(150-158), PRA(150-159), PRA(253-262) and PRA(254-262) are predicted to be made directly by an abundant cleavage site of Nardilysin. The latter two peptides (PRA(253-262), and PRA(254-262)) were, in addition, experimentally shown to be generated by a proteasomal cleavage at their C-terminus.
Example 3: Non-Proteasomal Cleavages are Required to Generate the C-Terminus of Proteasome-Independent HLA-A3-Presented CTL Epitope PRAME 190-198
(26) Some occasional CTL epitopes, mostly with a basic residue at their C-terminus, require non-proteasomal cleavages, by additional enzymes, to liberate their C-terminus (Tenzer et al., 2005; Cell. Mol. Life Sci 62:1025 and Seifert et al., 2003, Nat. Immunol. 4:375). The current invention includes one such a CTL epitope, position 190-198 in PRAME with aa. sequence ELFSYLIEK, of which the C-terminus is generated independently of the proteasome by two consecutive cleavages of Nardilysin (EC 3.4.24.61) and Thimet oligopeptidase (TOP; EC 3.4.24.15).
(27) In addition to its involvement in the production of the ELFSYLIEK epitope, Nardilysin was predicted to efficiently produce by a direct cleavage the C-termini of the HLA-A3 binding peptide PRA.sup.16-24 (SMSVWTSPR), the HLA-A68 binding peptides PRA.sup.150-158 and PRA.sup.150-159 (EAAQPMTKK and EAAQPMTKKR), the HLA-A24 binding peptide PRA.sup.254-262 and the HLA-B*3501 binding peptide PRA.sup.253-262. The latter two peptides (PRA.sup.254-262 and PRA.sup.253-262) were C-terminally also made by a proteasomal cleavage (as indicated in table 4).
(28) Material and Methods and Results of Determination of Enzymatic Generation of the N-Terminus and C-Terminus of PRAME.sup.190-198
(29) Purified preparations of Proteasome, Nardilysin and Thimet oligopeptidase (TOP), at a concentration of 20 nM, were used to digest in a cell free system synthetic 27-mer (PRA.sup.182-208), 19-mer (PRA.sup.190-208), 13-mer (PRA.sup.190-202), 12-mer (PRA.sup.190-201) and 11-mer (PRA.sup.190-200) peptides (at a concentration of 20 uM) encompassing the HLA-A3 presented CTL epitope ELFSYLIEK (PRA.sup.190-198) with its natural flanking regions. As summarized in
(30) In addition, functional recognition experiments using the CTL clone recognizing the ELFSYLIEK epitope (see
(31) Because of the closeness of the binding motif of HLA-A3 to that of HLA-A11, this novel epitope is also claimed as a novel epitope presented by HLA-A11. Target cells expressing HLA-A11 and PRAME were specifically recognized by the CTL anti-ELFSYLIEK (data not shown).
Example 4: Determination of Immunogenicity and Endogenous Production of the Identified CTL Epitopes Lymphocytes
(32) The analysis of the immunogenicity was performed for a subset of the identified putative HLA class I presented CTL epitopes. Immunogenicity was determined by in vitro inductions of CTL against the synthetically produced CTL epitopes. Moreover, the CTL (clones) that were generated have been tested for their capacity to recognize tumor cells co-expressing PRAME and the correct HLA class I molecule.
(33) CTL bulk cultures were induced against the following selected HLA class I binding PRAME derived CTL epitopes. The peptides PRA.sup.100-108 (VLDGLDVLL), PRA.sup.142-151 (SLYSFPEPEA), PRA.sup.300-309 (ALYVDSLFFL), PRA.sup.371-380 (ALLERASATL), and PRA.sup.425-433 (SLLQHLIGL) were chosen because these peptides are predicted CTL epitopes presented in HLA-A2. Furthermore, CTL were induced against PRA.sup.190-198 (ELFSYLIEK), which is a CTL epitope presented in HLA-A3, PRA.sup.113-122 (RPRRWKLQVL), which is an HLA-B7 presented epitope, and PRA.sup.258-267 (QMINLRRLLL), which is predicted to be an HLA-B8 expressed CTL epitope.
(34) Procedure of In Vitro Generation of CTL Clones and Functional CTL Assays
(35) Peripheral blood mononuclear cells (PBMC) for CTL inductions were obtained by the Ficoll-Paque method from blood from healthy donors. To optimally use all APC present in PBMC we developed a culture system that yields a mix of activated B cells and mature DC to be used as APC during the primary induction step. PBMC were separated in a T cell fraction and a fraction containing B cells and monocytes by SRBC-rosetting. The T cell fraction was cryopreserved. The mixture of monocytes and B cells was cultured in 24 wells plates at a concentration of 1?10.sup.6 cells/well in complete culture medium containing 800 U/ml GM-CSF, 500 U/ml IL-4 (PeproTech Inc.) and 500 ng/ml CD40 mAb (clone B-B20; Serotec) for 6 days. This culture system achieved a threefold effect: i) GM-CSF and IL-4 induced differentiation of monocytes into immature dendritic cells, ii) IL-4 and CD40 mAb caused activation and proliferation of B cells (Schultze, et al. 1997, J Clin. Invest. 100:2757) and iii) CD40 mAb mediated maturation of immature dendritic cells (Cella, et al. 1996. J Exp Med 184:747). At day 3, cytokines and CD40 mAb were replenished. To further promote CTL inducing capacity, the APC-mix was cultured for an additional 2 days with 0.4 ng/ml LPS (Difco Labs), 500 U/ml IFN (Boehringer Mannheim) and 500 ng/ml CD40 mAb. At day 8 the APC-mix was pulsed with 50 ?g/ml peptide (each peptide separately) for 4 h at RT, irradiated (30 Gy) and washed to remove free peptide. The cryopreserved autologous T cell fraction was thawed and depleted from CD4 T cells using magnetic beads (Dynal). The primary induction was performed in 96 well U-bottom plates. APC at a concentration of 10,000/well were co-cultured with 50,000 CD8.sup.? T cells/well in culture medium, containing 10% human pooled serum (HPS), 5 ng/ml IL-7 (PeproTech) and 0.1 ng/ml IL-12 (Sigma). At day 7 after initiation of induction the CTL micro-cultures were harvested (pooled), washed and restimulated at a concentration of 40,000 responder cells/well of 96-well U-bottom plates in culture medium containing 10% HPS, 5 ng/ml IL-7 and 0.1 ng/ml IL-12. Autologous activated B cells, generated via the protocol described by Schultze et al. (1997, J Clin. Invest. 100:2757), irradiated (75 Gy) and peptide pulsed (50 ?g/ml) for 4 h at RT in culture medium containing 2% FCS and 3 ?g/ml ?.sub.2-microglublin (Sigma) after mild acid elution to remove naturally presented peptides from the MHC I molecules (see material and methods MHC binding assay), were used at a concentration of 10,000 cells/well as restimulator APC. Restimulations were repeated at day 14 and 21 in a similar way, with the exception of IL-7 being replaced by 20 IU/ml 1L-2 (Chiron Corp.). At day 29, the CTL bulk culture was cloned by standard limiting dilution procedures. CTL clones were maintained by aspecific stimulation every 7 to 12 days using a feeder mixture consisting of allogeneic PBMC and B-LCL in culture medium containing 10% FCS, 1.5% leucoagglutinin (Sigma) and 240 IU/ml IL-2.
(36) For functional analysis of CTL capacity to kill peptide loaded target cells or tumor target cells a standard chromium release assays was used. After .sup.51Cr labeling (1 h), target cells (2000/well) were added to various numbers of effector cells in a final volume of 100 ?l complete culture medium in 96-well-U-bottom plates. After 4 h incubation at 37? C. supernatants were harvested. The mean % specific lysis of triplicate wells was calculated according to: (Experimental release?Spontaneous release)/(Maximal release?Spontaneous release)?100%.
(37) Results of the Analysis of Immunogenicity and Functional Recognition of Tumor Cells by CTL.
(38) The 8 peptides that were chosen for in vitro CTL inductions, which are PRA.sup.100-108 (HLA-A2), PRA.sup.142-151 (HLA-A2), PRA.sup.300-309 (HLA-A2), PRA.sup.371-380 (HLA-A2), PRA.sup.425-433 (HLA-A2), PRA.sup.190-198 (HLA-A3), PRA.sup.113-122 (HLA-B7) and PRA.sup.258-267 (HLA-B8), were all capable to induce bulk CTL cultures that highly specifically recognized the inducing peptide when loaded in the correct HLA class I molecule expressed on B-LCL target cells (data not shown). Subsequently, these CTL bulk cultures were cloned by limiting dilution, and CTL clones were generated.
(39) The CTL clones efficiently recognized the CTL epitopes against which they were raised, either as exogenously loaded synthetic peptide (
Example 5: Determination of CD4+ T Helper Cell Reactivity Against HLA Class II Binding Peptides in PRAME
(40) For the optimal induction and maintenance of a vaccine induced anti-tumor CD8.sup.+ CTL response, capable of eradication of PRAME expressing tumor cells, the induction of a concurrent CD4.sup.+ Th response is required (e.g. Bourgeois, et al, 2002. Eur. J. Immunol. 32:2199; Kumaraguru, et al, 2004. J. Immunol. 172:3719; Janssen, et al, 2003. Nature 421:852; Hamilton, et al, 2004. Nat. Immunol. 5:873). The primary mechanism contributing to this phenomenon is the help provided by the CD4.sup.+ helper T cell population in the maturation of professional antigen presenting cellsmainly dendritic cells (DCs)via the CD40-ligand CD40 interaction, which is termed the licensing model (Schoenberger, et al., 1998. Nature 393:480; Lanzavecchia. 1998. Nature 393:413). Several lines of evidence have shown that without such a CD4.sup.+ Th response the CD8.sup.+ response is not or only suboptimal induced and the maintenance and recall of the memory CD8.sup.+ T cell response is compromised (Belz, et al., 2002. J. Virol. 76:12388). It is crucial, therefore, to identify the HLA class II binding peptides in the PRAME protein that are capable of inducing CD4.sup.+ Th cells. These PRAME peptides were identified using two different screening assays. Both CD4.sup.+ Th cell proliferation and IFN? produced by Th cells were used to assess the reactivity against a panel of 51 overlapping PRAME peptides with a length needed for HLA class II binding (22-mer or 27-mer peptides). First, the HLA class II molecules that have predicted binding capacity for these overlapping PRAME peptides were identified.
(41) In Silico Determination of HLA Class II Binding Profile of Overlapping Polypeptides (27-Mer or 22-Mer) Derived from PRAME
(42) HLA class II peptide binding is less stringent than HLA class I binding. Peptides binding in HLA class II are at least 13 aa. long and may be much longer because the open end of the HLA class II binding groove allows peptides bound to class II molecules to extend beyond the groove at both ends. Therefore, length requirements of HLA class II binding peptides are much more flexible than the requirements of peptides binding in HLA class I molecules. Furthermore, and in line with this, peptide binding in HLA class II is more promiscuous than binding in HLA class I. Often a polypeptide of a length of 13 to 25 aa. has the capacity to bind in multiple HLA class II molecules. The advantage of these flexible peptide binding characteristics of HLA class II molecules is that actual experimental binding assays are much less needed to verify predicted peptide binding.
(43) For the prediction of HLA class II binding an algorithm that is freely available on the internet was used. This algorithm is ProPred (see Singh et al, 2001, Bioinformatics 17:1236). Using this algorithm, the 51 overlapping peptides were screened for the existence of binding motifs for the different HLA class II molecules and the results were analysed. As shown in Table 5A, all the overlapping peptides that were tested for CD4.sup.+ T cell reactivity had a predicted efficient binding capacity for multiple HLA class II molecules (cutoff used: the five predicted best binding peptides from full length PRAME for each class II allele).
(44) TABLE-US-00006 TABLE 5A HLA class II binding capacity of 51 overlapping PRAME peptides Overlapping PRAME peptides (position and HLA class II molecules for which the peptide has predicted binding Pep. length) capacity (marked with the symbol X) No. Start End Length DR1 DR2 DR3 DR4 DR5 DR 7 DR8 DR 9 DR51 DR 52 DR53 DQ 2 DQ3 DQ4 1 1 27 27 X X X X X X X X X 2 15 36 22 X X X X X X X 3 19 45 27 X X X X X X X X X X 4 31 52 22 X X X X X X X X 5 37 63 27 X X X X X X X X X 6 48 69 22 X X X X 7 53 79 27 X X X X 8 66 87 22 X X X X X 9 70 96 27 X X X X X X 10 84 110 27 X X X X X X X 11 95 121 27 X X X X X X X X X X 12 98 124 27 X X X X X X X X X X X 13 110 131 22 X X X X X X X X X X 14 116 142 27 X X X X X X 15 124 145 22 X X X X X X X 16 133 159 27 X X X X X X 17 146 172 27 X X X X 18 158 184 27 X X X X X X 19 173 199 27 X X X X X 20 181 207 27 X X X X X X X 21 194 220 27 X X X X X X X X 22 205 231 27 X X X X X X X X X 23 217 238 22 X X X X X X X 24 222 248 27 X X X X X X X 25 234 255 22 X X X X X X 26 239 265 27 X X X X X X X X 27 247 273 27 X X X X X X X X X X 28 256 277 22 X X X X X X X X X X 29 262 288 27 X X X X X X X X 30 276 302 27 X X X X X X X X X 31 290 316 27 X X X X X X X X X X X 32 300 326 27 X X X X X X 33 311 337 27 X X X X X X 34 323 349 27 X X X X X X 35 333 354 22 X X X X X X X 36 338 364 27 X X X X X X X X X 37 353 379 27 X X X X X X X X X 38 359 385 27 X X X X X X X X 39 372 398 27 X X X X X X 40 384 410 27 X X X X X 41 395 416 22 X X X X 42 399 425 27 X X X X X X X X X 43 412 433 22 X X X X X X X X X X X 44 415 441 27 X X X X X X X X X X X 45 424 450 27 X X X X X X X 46 434 455 22 X X X 47 442 463 22 X X X X 48 447 473 27 X X X X X X X X X 49 460 486 27 X X X X X X 50 473 499 27 X X X X X 51 483 509 27 X X
Procedure for CD4.sup.+ T Cell Proliferation Assay and CD4.sup.+ T Cell IFN? ELISPOT Assay
(45) For the CD4.sup.+ T cell proliferation assay, total PBMC (1.5?10e5 cells/well), either obtained from healthy donors or patients with a PRAME-positive cancer, were seeded in 8 wells of a U-bottom 96-wells plate in RPMI culture medium supplemented with 10% autologous serum and 10 ?g/ml of 51 overlapping 27-mer or 22-mer PRAME peptides. At day 6, 50 ?l of 3H-thymidine (1 mCi/50 ml) was added and at day 7 the incorporation of 3H-thymidine was measured.
(46) For the IFN? ELISPOT assay, CD45RO.sup.+ cells were isolated from PBMC using CD45RO magnetic beads from Miltenyi Biotec. Subsequently, CD45RO.sup.+ (and CD45RO-negative) cells were seeded in 10 wells of a 24-wells plate (2-3?10e6 cells/well) together with autologous irradiated PBMC at a ratio of 4:1 in IMDM with 10% human pooled serum supplemented with 10 peptide mixes of 5 different peptides each from the panel of 51 overlapping 27-mer or 22-mer PRAME peptides. The peptide concentration of each peptide was 5 ?g/ml, and IL-2 (150 IU/ml) was added at day 2. At day 10, the peptide-stimulated CD45RO cultures were counted and seeded in IFN? ELISPOT plates together with autologous irradiated PBMC at a ratio of 1:1 in triplicate in the absence of peptide or in the presence of 5 ?g/ml of the separate peptides no 1 to no 51.
(47) Results of CD4.sup.+ T Cell Reactivity Against the Panel of 51 PRAME 27-Mer/22-Mer Peptides
(48) The analysis of CD4.sup.+ Th cell reactivity against 51 overlapping PRAME peptides in peripheral blood of 8 healthy donors and 7 PRAME positive cancer patients, revealed that 28 out of the 51 peptides induced IFN? production by CD4.sup.+ Th cells and 36 peptides induced CD4.sup.+ Th cell proliferation (Table 5B).
(49) TABLE-US-00007 TABLE 5B Reactivity of 51 overlapping HLA class II binding PRAME peptides. IFN? produced by CD4.sup.+ Th cells Memory Naive fraction Memory fraction CD4.sup.+ Pept. position in fraction (in Th cell Pep. and length healthy in healthy prolifer- No. Start End Length donors patients donors ation 1 1 27 27 + + + 2 15 36 22 + + + 3 19 45 27 + + 4 31 52 22 + 5 37 63 27 + 6 48 69 22 + + 7 53 79 27 + + 8 66 87 22 + + + 9 70 96 27 + + + 10 84 110 27 + + + 11 95 121 27 + 12 98 124 27 + + 13 110 131 22 + + + + 14 116 142 27 + + 15 124 145 22 + 16 133 159 27 + + 17 146 172 27 18 158 184 27 19 173 199 27 + 20 181 207 27 + + + 21 194 220 27 + + 22 205 231 27 + + + 23 217 238 22 + 24 222 248 27 + + 25 234 255 22 + + 26 239 265 27 + 27 247 273 27 + + 28 256 277 22 + + + 29 262 288 27 + + 30 276 302 27 + 31 290 316 27 + 32 300 326 27 + 33 311 337 27 + 34 323 349 27 + 35 333 354 22 + 36 338 364 27 37 353 379 27 + + 38 359 385 27 39 372 398 27 40 384 410 27 41 395 416 22 + 42 399 425 27 + + 43 412 433 22 + 44 415 441 27 45 424 450 27 + + 46 434 455 22 + 47 442 463 22 48 447 473 27 + + 49 460 486 27 + + 50 473 499 27 51 483 509 27 +
Example 6: Selection of Vaccine Peptides Fulfilling the Major Vaccine Requirements
(50) An optimal and defined T cell-inducing composition, comprising one or more PRAME derived peptides, inducing an immune response against PRAME positive tumors must induce both an HLA class I restricted CD8.sup.+ CTL response and, simultaneously, an HLA class II restricted CD4.sup.+ T helper response. The Th cell response is required to enhance the induction and to maintain the CTL response.
(51) Moreover, due to the extensive polymorphism of the HLA molecules, an optimal vaccine needs to be designed in order to have a broad HLA haplotype coverage allowing a use of this vaccine for a large potential population of subjects. Furthermore, the vaccine should be suitable for a high percentage of individual patients with PRAME positive cancers. Therefore, a vaccine composition according to this invention contains multiple PRAME CTL epitopes that are presented in different HLA class I molecules with a high prevalence in the population. Because of the high degree of promiscuous binding in HLA class II molecules, this requirement is less strictly required for CD4.sup.+ T helper cell inducing peptides. The identification of CTL epitopes, as summarized in Table 4, and CD4.sup.+ T helper epitopes, as listed above in Table 5A and 5B, enabled the design of vaccine peptides to be contained in a defined vaccine for PRAME positive cancers.
(52) The vaccine composition comprises PRAME derived peptides of 30-35 aa. in length, because several advantages are associated with peptides of this size. As mentioned before, such peptides are in principle easy to synthesize. Furthermore, they have sufficient length to contain both HLA class I presented CTL epitopes and HLA class II presented T helper epitopes. Finally, of great importance is that peptides of this length need to be processed by professional antigen presenting cells, in particular dendritic cells, before the epitopes (both CTL and T helper) can be presented by the antigen presenting cell (Zwaveling, et al, 2002. J. Immunol. 169:350). As a consequence, presentation on non-professional antigen presenting cells and systemic spread through the organism will not take place, and therefore, the induction of tolerance, which has been observed after vaccination with minimal HLA class I presented CTL epitopes (Toes, et al, 1996. J. Immunol. 156:3911; Toes, et al, 1996. Proc. Natl. Acad. Sci. U.S.A 93:7855), will not occur. Therefore, vaccine peptides of this length are superior over short minimal HLA class I epitopes or full length proteins.
(53) Using the information of the identified CD8.sup.+ CTL epitopes and CD4.sup.+ T helper reactive PRAME derived peptides, 20 PRAME vaccine peptides were designed that comply with the following three major rules: 1) containing at least one CTL epitope, preferably more than one, and most preferably also CTL epitopes of which the immunogenicity was confirmed by CTL inductions and more preferably presentable by HLA-A2, 2) containing at least one CD4.sup.+ T helper cell reactive peptide, preferably reactive both in patients having a PRAME positive malignancy and in healthy donors and 3) a length of 19-45 aa., preferably 30 to 35 amino acids.
(54) The PRAME derived peptides listed in Table 6, are designed according to this invention and fulfil to these requirements. The PRAME derived peptides in Table 6 have a superior capacity to mount an effective, enhanced and prolonged immune response against PRAME expressing malignancies and tumors in human subjects in vivo than PRAME fragments and compositions previously described in the art.
(55) Each of the peptides of the invention as listed in Table 6 has actually been synthesized and purified as described in Example 1 herein above. However, for one peptide (SEQ ID NO. 22: amino acids 222-256 of SEQ ID NO. 21), that was initially designed using the same criteria as for the peptides in Table 6, we found that in practice it could not be synthesized in acceptable purity (less than 2% correct sequence). We further note that each of these peptides of the invention is soluble in physiologically acceptable salt solutions (comprising at most 35% DMSO) at concentrations in the range of 0.5-8 mg/ml.
(56) TABLE-US-00008 TABLE6 TwentyPRAMEvaccinepeptides(IDNo's1-20;length33-35aa.)andtheircharacterizationwith respecttoHLAclassIandHLAclass HLAclassII epitopes containedin HLAclassIepitopescontainedin Vaccine vaccinepeptide vaccinepeptide peptide.sup.a HLA HLAclassI (No.and HLA CD4.sup.+- bindingcapacity position classII Thcell HLA Pro- Intact in Binding Reacti- HLAclassIbindingpeptide ClassI Binding cessing Frag- (PRAME Peptide.sup.b vity.sup.c Start End Sequence.sup.e Length.sup.f allele (IC.sub.50.sup.g) C-term.sup.h ment.sup.i CTL.sup.j #1 1-27 IFN?/ 5 14 RLWGSIQSRY 10 HLA-A3 1.59 - - n.t. PRAME Prolif 5 13 RLWGSIQSR 9 HLA-A3 1.13 - - n.t. 1-33 13 22 RYISMSVWTS 10 HLA-A24 5.8 - - n.t. 16 24 SMSVWTSPR 9 HLA-A3 <1 +.sup.(k) NT n.t. #2 19-45 IFN?/ 25 34 RLVELAGQSL 10 HLA-A2 11.1 - - n.t. PRAME Prolif 28 36 ELAGQSLLK 9 HLA-A3 3.14 - - n.t. 19-53 33 42 SLLKDEALAI 10 HLA-A2 14.0 ++ + n.t. 34 42 LLKDEALAI 9 HLA-A2 10.2 ++ + n.t. 36 45 KDEALAIAAL 10 HLA-B60 2.91 ++ ND n.t. 37 45 DEALAIAAL 9 HLA-B60 1.55 ++ ND n.t. 37 45 DEALAIAAL 9 HLA-B61 <1 ++ ND n.t. 39 47 ALAIAALEL 9 HLA-A2 5.1 - - n.t. 39 48 ALAIAALELL 10 HLA-A2 9.0 - - n.t. 41 50 AIAALELLPR 10 HLA-A3 10.75 - - n.t. #3 48-69 IFN?/ 47 56 LLPRELFPPL 10 HLA-A2 2.1 - - - PRAME Prolif 53-79 IFN?/ 48 56 LPRELFPPL 9 HLA-B*3501 <1 - - n.t. Prolif 48 57 LPREIFPPLF 10 HLA-B*3501 1.58 + ND n.t. 50 58 RELFPPLFM 9 HLA-B60 1.48 ++ + n.t. 50 58 RELFPPLFM 9 HLA-B61 <1 ++ + n.t. 50 59 RELFPPLFMA 10 HLA-B61 <1 ++ + n.t. 52 61 LFPPLFMAAF 10 HLA-A24 <1 ++ ND n.t. 53 61 FPPLFMAAF 9 HLA-B*3501 <1 - ND n.t. 60 69 AFDGRHSQTL 10 HLA-A24 5.5 + + n.t. #4 70-96 IFN?/ 71 80 AMVQAWPFTC 10 HLA-A2 10.4 - - n.t. PRAME Prolif 77 86 PFTCLPLGVL 10 HLA-A24 2.1 ++ + n.t. 69-101 80 88 CLPLGVLMK 9 HLA-A3 <1 - - n.t 85 94 VLMKGQHLHL 10 HLA-A24 15 - - n.t. 89 97 GQHLHLETF 9 HLA-B62 2.39 ++ + n.t. 91 99 HLHLETFKA 9 HLA-A2 11.1 - - n.t 94 101 LETFKAVL 8 HLA-B61 <1 ++ + n.t #5 84-110 IFN?/ 80 88 CLPLGVLMK 9 HLA-A3 <1 - - n.t. PRAME Prolif 85 94 VLMKGQHLHL 10 HLA-A24 15 - - n.t. 80-114 89 97 GQHLHLETF 9 HLA-B62 2.39 ++ + n.t. 91 99 HLHLETFKA 9 HLA-A2 11.1 - - n.t. 94 101 LETFKAVL 8 HLA-B61 <1 ++ + n.t. 96 104 TFKAVLDGL 9 HLA-A24 8.6 - - n.t. 99 108 AVLDGLDVLL 10 HLA-A2 9.4 ++ + n.t. 99 107 AVLDGLDVL 9 HLA-A2 13.4 - - n.t. 100 108 VLDGLDVLL 9 HLA-A2 5.2 ++ + + 100 109 VLDGLDVLLA 10 HLA-A2 11.9 - - n.t. 103 112 GLDVLLAQEV 10 HLA-A2 15.2 - - n.t. #6 98-124 IFN?/ 94 101 LETFKAVL 8 HLA-B61 <1 + + n.t PRAME Prolif 96 104 TFKAVLDGL 9 HLA-A24 8.6 - - n.t. 94-126 99 107 AVLDGLDVL 9 HLA-A2 13.4 - - n.t. 99 108 AVLDGLDVLL 10 HLA-A2 9.4 ++ + n.t. 100 108 VLDGLDVLL 9 HLA-A2 5.2 ++ + + 100 109 VLDGLDVLLA 10 HLA-A2 11.9 - - n.t. 103 112 GLDVLLAQEV 10 HLA-A2 15.2 - - n.t. 107 116 LLAQEVRPRR 10 HLA-A3 14.0 - - n.t. 113 122 RPRRWKLQVL 10 HLA-B7 <1 + + + 113 122 RPRRWKLQVL 10 HLA-B8 <1 + + n.t. 118 126 KLQVLDLRK 9 HLA-A3 2.15 - - n.t. #7 116-142 IFN?/ 113 122 RPRRWKLQVL 10 HLA-B7 <1 + + + PRAME Prolif 113 122 RPRRWKLQVL 10 HLA-B8 <1 + + n.t. 112-144 118 126 KLQVLDLRK 9 HLA-A3 2.15 - - n.t. 136 144 WSGNRASLY 9 HLA-A1 4.3 - - n.t. #8 133-159 IFN?/ 136 144 WSGNRASLY 9 HLA-A1 4.3 - - n.t. PRAME Prolif 142 151 SLYSFPEPEA 10 HLA-A2 1.9 ++ + + 133-166 150 158 EAAQPMTKK 9 HLA-A*6801 Pred. +.sup.(k) NT n.t. 150 159 EAAQPMTKKR 10 HLA-A*6801 Pred. +.sup.(k) NT n.t. #9 181-207 IFN?/ 173 182 IPVEVLVDLF 10 HLA-A24 <1 - - n.t. PRAME Prolif 182 191 FLKEGACDEL 10 HLA-A2 3.0 - - n.t. 173-207 186 195 GACDELFSYL 10 HLA-A2 10.6 - - n.t. 186 194 GACDELFSY 9 HLA-B*3501 2.60 + - n.t. 190 199 ELFSYLIEKV 10 HLA-A2 4.5 - - n.t. 190 198 ELFSYLIEK 9 HLA-A3 1.42 +.sup.(k) + + 194 202 YLIEKVKRK 9 HLA-A3 3.49 - - n.t. 194 203 YLIEKVKRKK 10 HLA-A3 14.0 - - n.t. 198 207 KVKRKKNVLR 10 HLA-A3 7.5 - - n.t. #10 194-220 IFN? 190 199 ELFSYLIEKV 10 HLA-A2 4.5 - - n.t. PRAME 190 198 ELFSYLIEK 9 HLA-A3 1.42 +.sup.(k) + + 190-223 194 202 YLIEKVKRK 9 HLA-A3 3.49 - - n.t. 194 203 YLIEKVKRKK 10 HLA-A3 14.0 - - n.t. 198 207 KVKRKKNVLR 10 HLA-A3 7.5 - - n.t. 204 212 NVLRLCCKK 9 HLA-A3 13.5 - - n.t. 205 214 VLRLCCKKLK 10 HLA-A3 1.3 - - n.t. 214 223 KIFAMPMQDI 10 HLA-A2 7.2 - - n.t. 215 223 IFAMPMQDI 9 HLA-A24 1.8 - - n.t. #11 234-255 IFN?/ 242 250 CTWKLPTLA 9 HLA-A2 9.3 - - n.t. PRAME Prolif 242 251 CIANKLPTLAK 10 HLA-A3 0.7 - - n.t. 234-268 246 255 LPTLAKFSPY 10 HLA-B*3501 0.11 - - n.t. 247 255 PTLAKFSPY 9 HLA-A1 8.5 - - n.t. 248 256 TLAKFSPYL 9 HLA-A2 4.6 + + n.t. 251 260 KFSPYLGQMI 10 HLA-A24 2.5 - - n.t. 253 262 SPYLGQMINL 10 HLA-B*3501 1.98 +.sup.(k) + n.t. 254 262 PYLGQMINL 9 HLA-A24 <1 + n.t. 255 264 YLGQMINLRR 10 HLA-A3 4.5 - - n.t. 258 267 QMINLRRLLL 10 HLA-A2 4.0 + + n.t. 258 267 QMINLRRLLL 10 HLA-B8 1.67 + + + 259 266 MINLRRLL 8 HLA-B8 <1 + + n.t. 260 267 INLRRLLL 8 HLA-B8 <1 + + n.t. #12 247-273 IFN?/ 248 256 TLAKFSPYL 9 HLA-A2 4.6 + + n.t. PRAME Prolif 247-279 256-277 IFN?/ 251 260 KFSPYLGQMI 10 HLA-A24 2.5 - - n.t. Prolif 253 262 SPYLGQMINL 10 HLA-B*3501 1.98 +.sup.(k) + n.t. 254 262 PYLGQMINL 9 HLA-A24 <1 +.sup.(k) + n.t. 255 264 YLGQMINLRR 10 HLA-A3 4.5 - - n.t. 258 267 QMINLRRLLL 10 HLA-A2 4.0 + + n.t. 258 267 QMINLRRLLL 10 HLA-B8 1.67 + + + 259 266 MINLRRLL 8 HLA-B8 <1 + + n.t. 260 267 INLRRLLL 8 HLA-B8 <1 + + n.t. 261 269 NLRRLLLSH 9 HLA-A3 3.5 - - n.t. 267 275 LSHIHASSY 9 HLA-A1 1.0 - - n.t. #13 262-288 IFN? 267 275 LSHIHASSY 9 HLA-A1 1.0 - - n.t. PRAME 275 284 YISPEKEEQY 10 HLA-A1 3.0 - - n.t. 262-294 283 292 QYIAQFTSQF 10 HLA-A24 8.2 ++ + n.t. 284 293 YIAQFTSQFL 10 HLA-A2 10.4 ++ + n.t. #14 290-316 Prolif. 284 293 YIAQFTSQFL 10 HLA-A2 10.4 ++ + n.t. PRAME 287 295 QFTSQFLSL 9 HLA-A24 1.0 ++ ND n.t. 284-316 292 301 FLSLQCLQAL 10 HLA-A2 2.5 - - n.t. 292 302 FLSLQCLQALY 11 HLA-A1 1.0 - - n.t. 293 302 LSLQCLQALY 10 HLA-A1 2.9 - - n.t. 294 302 SLQCLQALY 9 HLA-A1 2.0 - - n.t. 294 303 SLQCLQALYV 10 HLA-A2 3.2 + - n.t. 300 308 ALYVDSLFF 9 HLA-A2 2.7 + + n.t. 300 308 ALYVDSLFF 9 HLA-A3 8 + + n.t. 300 308 ALYVDSLFF 9 HLA-B62 <1 + + n.t. 300 309 ALYVDSLFFL 10 HLA-A2 1.7 ++ + + 301 309 LYVDSLFFL 9 HLA-A2 6.3 ++ + n.t. 301 309 LYVDSLFFL 9 HLA-A24 <1 ++ + n.t. 302 310 YVDSLFFLR 9 HLA-A1 1.4 + - n.t. 302 310 YVDSLFFLR 9 HLA-A*6801 <1 + - n.t. 307 316 FFLRGRLDQL 10 HLA-A24 1.8 - - n.t. #15 300-326 Prolif. 300 308 ALYVDSLFF 9 HLA-A2 2.7 + + n.t. PRAME 300 308 ALYVDSLFF 9 HLA-A3 8 + + n.t. 295-327 300 308 ALYVDSLFF 9 HLA-B62 <1 + + n.t. 300 309 ALYVDSLFFL 10 HLA-A2 1.7 ++ + + 301 309 LYVDSLFFL 9 HLA-A2 6.3 ++ + n.t. 301 309 LYVDSLFFL 9 HLA-A24 <1 ++ + n.t. 302 310 YVDSLFFLR 9 HLA-A1 1.4 + - n.t. 302 310 YVDSLFFLR 9 HLA-A*6801 <1 + - n.t. 307 316 FFLRGRLDQL 10 HLA-A24 1.8 - - n.t. 308 317 FLRGRLDQLL 10 HLA-A2 9.6 - - n.t. 316 324 LLRHVMNPL 9 HLA-B62 2.56 - - n.t. #16 353-379 IFN?/ 355 364 SLSGVMLTDV 10 HLA-A2 9.9 - - n.t PRAME Prolif 360 369 MLTDVSPEPL 10 HLA-A2 5.6 - - n.t. 353-387 361 370 LTDVSPEPLQ 10 HLA-A1 3.8 + + n.t. 361 371 LTDVSPEPLQA 11 HLA-A1 3.5 + + n.t. 371 380 ALLERASATL 10 HLA-A2 12.9 ++ + + #17 399-425 IFN?/ 405 414 CSQLTTLSFY 10 HLA-A1 <1 ++ - n.t. PRAME Prolif 410 418 TLSFYGNSI 9 HLA-A2 11.0 - - n.t. 399-431 412 420 SFYGNSISI 9 HLA-A24 <1 ++ + n.t. 419 427 SISALQSLL 9 HLA-A2 5.7 - - n.t. 422 431 ALQSLLQHLI 10 HLA-A2 3.2 - - n.t. 422 430 ALQSLLQHL 9 HLA-A2 14.2 - - n.t. #18 424-450 IFN?/ 419 427 SISALQSLL 9 HLA-A2 5.7 - - n.t. PRAME Prolif 422 431 ALQSLLQHLI 10 HLA-A2 3.2 - - n.t. 417-450 422 430 ALQSLLQHL 9 HLA-A2 14.2 - - n.t. 425 433 SLLQHLIGL 9 HLA-A2 3.7 ++ + + 427 436 LQHLIGLSNL 10 HLA-B62 2.41 + + n.t. 429 438 HLIGLSNLTH 10 HLA-A3 4.0 + + n.t. 432 440 GLSNLTHVL 9 HLA-A2 6.8 + - n.t. 432 441 GLSNITHVLY 10 HLA-A3 4.07 - - n.t. 433 441 LSNITHVLY 9 HLA-A1 <1 - - n.t. 435 443 NLTHVLYPV 9 HLA-A2 2.5 - - n.t. 439 448 VLYPVPLESY 10 HLA-A1 10.9 + + n.t. 439 448 VLYPVPLESY 10 HLA-A3 2.67 + + n.t. 439 448 VLYPVPLESY 10 HLA-B62 1.66 + + n.t. #19 447-473 IFN?/ 447 455 SYEDIHGTL 9 HLA-A24 <1 - - n.t. PRAME Prolif 448 457 YEDIHGTLHL 10 HLA-B60 <1 ++ - n.t. 447-480 453 462 GTLHLERLAY 10 HLA-A1 2.0 - - n.t. 454 462 TLHLERLAY 9 HLA-A1 10.1 - - n.t. 454 463 TLHLERLAYL 10 HLA-A2 12.2 - - n.t. 459 467 RLAYLHARL 9 HLA-A24 <1 ++ + n.t. 459 468 RLAYLHARLR 10 HLA-A3 1.0 - - n.t. 461 470 AYLHARLREL 10 HLA-A24 1 + + n.t. 462 470 YLHARLREL 9 HLA-A2 6.2 + + n.t. 462 470 YLHARLREL 9 HLA-B8 <1 + + n.t. 462 471 YLHARLRELL 10 HLA-A2 13.3 + + n.t. 466 474 RLRELLCEL 9 HLA-A2 14.0 - - n.t. 466 474 RLRELLCEL 9 HLA-A24 <1 - - n.t. 470 479 LLCELGRPSM 10 HLA-A2 10.5 - - n.t. #20 483-509 IFN? 487 496 CPHCGDRTFY 10 HLA-B*3501 1.5 - - n.t. PRAME 494 502 TFYDPEPIL 9 HLA-A24 <1 - - n.t. 477-509 499 507 EPILCPCFM 9 HLA-B*3501 0.32 - - n.t. .sup.aVaccine peptides of 33 to 35 aa. length: peptide ID No. and positions of first and last aa. in full length PRAME protein. .sup.bStart and end position (aa.) of the HLA class II binding peptide that was tested for CD4. Th cell reactivity. .sup.cCD44 Th cell reactivity against HLA class II binding peptides. Nomenclature: IFN?: IFN?-response observed after stimulation with the indicated peptide; Prolif.: Proliferative response observed after stimulation with indicated peptide. .sup.dPostion in PRAME of the N-terminal amino acid of the HLA class I binding peptide. Peptides are sorted by the starting aa. .sup.eAa. sequence of the HLA class I binding peptide .sup.fLength of the HLA class I binding peptide .sup.gIC.sub.50 is the peptide concentration needed to inhibit binding of FL-labeled reference peptide for 50% (IC.sub.50 in mM). Pred., predicted high binding affinity. .sup.hGeneration by proteasome-mediated digestion of fragments containing the correct C-terminus of the HLA class I binding peptide. Digestion was assessed at 1 h digestion because this is physiologically the most relevant time point. Classification: (++) fragments present for >5%, (+) present for <5%, (-) no fragments containing the C-term. were found. Peptides with IC.sub.50 <15 mM are considered to be potential CTL epitopes with respect to their binding affinity. .sup.iIntact epitope found in digestion fragments after 1 h digestion: (+), present; (-), not present; (ND), could not be determined due to artificial ends of the synthetic input peptides; (NT), intactness of these epitopes after digestion with nardilysin was not tested.? .sup.jCTL induced against this specific HLA/peptide combination, specifically recognizing tumor cells. Classification: +, CTL induced and recognize tumor cells; -, CTL induced but do not recognize tumor cells; n.t., not tested? .sup.kHLA-A3 presented CTL epitope PRA(190-198) (ELFSYLIEK) is generated by non-proteasomal cleavages as explained in Example 3 and FIG. 2. The C-termini of PRA(16-24), PRA(150-158), PRA(150-159), PRA(253-262) and PRA(254-262) are predicted to be made directly by an abundant cleavage site of Nardilysin. The latter two peptides (PRA(253-262), and PRA(254-262)) were, in addition, experimentally shown to be generated by a proteasomal cleavage at their C-terminus (see table 4).?