Survivin-derived peptides and uses thereof
09534030 ยท 2017-01-03
Assignee
Inventors
Cpc classification
A61K47/00
HUMAN NECESSITIES
C07K14/4738
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
A61P1/18
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
A61K47/51
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
G01N33/57484
PHYSICS
A61K39/39
HUMAN NECESSITIES
A61K39/00115
HUMAN NECESSITIES
A61K40/424
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
International classification
A61K47/00
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
Abstract
MHC Class I-restricted peptides derived from the tumor associated antigen, survivin, which peptides are capable of binding to Class I HLA molecules at a high affinity, capable of eliciting INF--producing cells in a PBL population of a cancer patient and capable of in situ detection of cytotoxic T cells in a tumor tissue, therapeutic and diagnostic composition comprising the peptide and uses thereof.
Claims
1. An immunogenic composition consisting of the peptide of the amino acid sequence of SEQ ID NO: 18 and an adjuvant.
2. An immunogenic composition consisting of the peptide of the amino acid sequence of SEQ ID NO: 18 coupled to an immunogenic carrier, and an adjuvant.
3. An immunogenic composition consisting of the peptide of the amino acid sequence of SEQ ID NO: 18, an adjuvant, and at least one of a pharmaceutical carrier, diluent, preservative, buffer component, or a combination thereof.
Description
(1) The invention will now be described in further details in the below, non-limiting examples and the figures, wherein
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(19) In the following table, amino acid sequences for peptides used herein and their respective SEQ ID NOs are listed:
(20) TABLE-US-00002 SEQID NO: DESIGNATION SEQUENCE 1 Sur6 FLKLDRERA 2 Sur8 TLPPAWQPFL 3 Sur9 ELTLGEFLKL 4 Sur1L2 LLLGEFLKL 5 Sur1M2 LMLGEFLKL 6 Sur46-54 CPTENEPDL 7 Sur51-59 EPDLAQCFF 8 Sur46Y9 CPTENEPDY 9 sur51Y9 EPDLAQCFY 10 Sur1 LTLGEFLKL 11 C1 ILKEPVHGV 12 Sur2 RAIEQLAAM 13 Sur3 KVRRAIEQL 14 Sur4 STFKNWPFL 15 Sur5 SVKKQFEEL 16 Sur7 TAKKVRRAI 17 Sur10 ETAKKVRRAI 18 Sur6-14 LPPAWQPFL 19 Sur11-19 QPFLKDHRI 20 Sur34-43 TPERMAEAGF 21 C24 YPLHEQHQM 22 Sur14-22 LKDHRISTF 23 Sur38-46 MAEAGFIHC 24 Sur93-101 FEELTLGEF 25 Sur47-56 PTENEPDLAQ 26 Sur49-58 ENEPDLAQCF 27 Sur92-101 QFEELTLGEF 28 C1 VSDGGPNLY 29 sur14Y9 LKDHRISTY 30 sur93Y9 FEELTLGEY 31 sur92Y9 QFEELTLGEY 32 sur34Y9 TPERMAEAGY 33 sur49Y9 ENEPDLAQCY 34 Sur92T2 QTEELTLGEF 35 Sur92S2 QSEELTLGEF 36 Sur93T2 FTELTLGEF 37 Sur93S2 FSELTLGEF 38 Sur38Y9 MAEAGFIHY 39 Sur47Y10 PTENEPDLAY 40 Sur5-13 TLPPAWQPF 41 Sur53-61 DLAQCFFCF 42 Sur54-62 LAQCFFCFK 43 Sur95-103 ELTLGEFLK 44 Sur112-120 KIAKETNNK 45 Sur13-22 FLKDHRISTF 47 Sur53-62 DLAQCFFCFK 50 Sur103-112 KLDRERAKNK 51 Sur112-121 KIAKETNNKK 52 Sur113-122 IAKETNNKKK 53 C3 ILRGSVAHK 54 Sur5K9 TLPPAWQPK 55 Sur53K9 DLAQCFFCK 56 Sur54L2 LLQCFFCFK 57 Sur13K9 FLKDHRISTK 58 Sur18K10 RISTFKNWPK 59 Sur113L2 ILKETNNKKK 60 SurEx3-A3-1 TIRRKNLRK 61 SurEx3-A3-2 PTIRRKNLRK 62 Sur2b-A3-1 RITREEHKK 63 C4 AVFDRKSDAK 64 C6 QPRAPIRPI 65 C7 RPPIFIRRL 66 Sur4-14 PTLPPAWQPFL 67 Sur18-28 RISTFKNWPFL 68 Sur54-64 LAQCFFCFKEL 69 Sur86-96 FLSVKKQFEEL 70 Sur88-98 SVKKQFEELTL 71 Sur103-113 KLDRERAKNKI 72 Ebv,BMLF1 GLCTLVAML 73 Hiv,Pol ILKEPVHGV 74 InfluenzaA, ILRGSVAHK nucleoprotein 265-273
Example 1
Identification of a Cytotoxic T-Lymphocyte Response to the Apoptosis Inhibitor Protein Survivin in Cancer Patients
(21) Summary
(22) Using CTL epitopes derived from survivin, specific T-cell reactivity against such antigens in peripheral blood from chronic lymphatic leukemia (CLL) patients and in tumor-infiltrated lymph nodes from melanoma patients by ELISPOT analysis have been studied. CTL responses to survivin-derived peptide epitopes were detected in three out of six melanoma patients and in three out of four CLL patients. No T-cell reactivity was detected in PBL from six healthy controls. Thus, survivin-derived peptides may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.
(23) Introduction
(24) The survivin protein was scanned for the presence of HLA-A*0201 (HLA-A2) binding peptide motifs and after successful identification, the peptides were used to test for specific T-cell reactivity in leukemia and melanoma patients by ELISPOT assay. In both patient cohorts CTL responses to two survivin-derived peptide epitopes were detected, whereas no T-cell reactivity could be detected in the healthy controls. These data suggest that survivin represent a widely expressed tumor antigen recognized by autologous T cells.
(25) Materials and Methods
(26) Patients and Normal Controls
(27) Peripheral vein blood samples from 4 patients diagnosed with CLL (designated CLL1-4) and blood samples from 6 normal individuals were collected into heparinised tubes. PBLs were isolated using LYMPHOPREP (density gradient medium) separation and frozen in fetal calf serum (FCS) with 10% di-methylsulphoxide. Additionally, T lymphocytes from tumor-infiltrated lymph nodes were obtained from 6 melanoma patients (designated mel1-6). Freshly resected lymph nodes were minced into small fragments, crushed to release cells into culture and cryopreserved. PBLs were available from 4 of the melanoma patients. All individuals included were HLA-A2 positive as determined by FACS analysis using the HLA-A2 specific antibody BB7.2. The antibody was purified from hybridoma supernatant. Patient samples were obtained from the State University Hospital, Herlev, Denmark. Informed consent was obtained from the patients prior to any of these measures.
(28) Survivin-Derived Peptides
(29) All peptides were obtained from Research Genetics (Huntsville, Ala., USA) and provided at >90% purity as verified by HPLC and MS analysis. The peptides used are listed in Table 1.
(30) TABLE-US-00003 TABLE1 PeptidesexaminedinthisstudyandtheirbindingaffinitytoHLA-A2 Name Protein.sup.a Sequence SEQIDNO: C.sub.50(M).sup.b C1 HIV-1pol.sub.476-484 ILKEPVHGV 11 0.7 Sur1 Survivin.sub.96-104 LTLGEFLKL 10 >100 Sur2 Survivin.sub.133-141 RAIEQLAAM 12 Notbinding Sur3 Survivin.sub.130-138 KVRRAIEQL 13 >100 Sur4 Survivin.sub.20-28 STFKNWPFL 14 Notbinding Sur5 Survivin.sub.88-96 SVKKQFEEL 15 Notbinding Sur6 Survivin.sub.101-109 FLKLDRERA 1 30 Sur7 Survivin.sub.127-135 TAKKVRRAI 16 Notbinding Sur8 Survivin.sub.5-14 TLPPAWQPFL 2 30 Sur9 Survivin.sub.95-104 ELTLGEFLKL 3 10 Sur10 Survivin.sub.126-135 ETAKKVRRAI 17 Notbinding Sur1L2 LLLGEFLKL 4 1 Sur1M2 LMLGEFLKL 5 1 .sup.aThe value range listed in subscript indicates the position of the peptide in the survivin sequence as disclosed in U.S. Pat. No. 6,245,523 .sup.bThe C.sub.50 value is the concentration of the peptide required for half maximal binding to HLA-A2 determined as described below
Assembly Assay for Peptide Binding to Class I MHC Molecules
(31) Assembly assays for binding of the synthetic peptides to class I MHC molecules metabolically labeled with [35S]-methionine were carried out as described (12,13). The assembly assay is based on stabilization of the class I molecules after loading of peptide to the peptide transporter deficient cell line T2. Subsequently, correctly folded stable MHC heavy chains are immunoprecipitated using conformation-dependent antibodies. After IEF electrophoresis, gels were exposed to phospholmager screens, and peptide binding was quantified using the IMAGEQUANT Phosphorlmager program (image analysis software) (Molecular Dynamics, Sunnyvale, Calif.).
(32) Antigen Stimulation of PBLs
(33) To extend the sensitivity of the ELISPOT assay, PBLs were stimulated once in vitro prior to analysis (14,15). Fresh and previously frozen PBLs gave similar results in the ELISPOT assay. On day 0, PBLs or crushed lymph node were thawed and plated in 2 ml/well at a concentration of 2106 cells in 24-well plates (Nunc, Denmark) in AIM V medium (Life Technologies, Roskilde, Denmark), 5% heat-inactivated human serum and 2 mM of L-glutamine in the presence of 10 M of peptide. In each experiment a well without peptide was included. Two days later 300 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT assay on day 12.
(34) ELISPOT Assay
(35) The ELISPOT assay used to quantify peptide epitope-specific interferon--releasing effector cells was performed as in (16). Briefly, nitrocellulose bottomed 96-well plates (MULTISCREEN MAIP N45 (96-well assay plate), Millipore, Hedehusene, Denmark) were coated with anti-IFN- antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by AIM V medium (serum-free media), and cells were added in duplicates at different cell concentrations. Peptides were then added to each well and the plates were incubated overnight. On the following day, medium was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed and Avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies) was added to each well. Plates were incubated at RT for 1 hour and the enzyme substrate NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate) (Gibco, Life Technologies) was added to each well and incubated at room temperature for 5-10 min. The reaction was terminated by washing with tap water upon the emergence of dark purple spots. The spots were counted using the ALPHA IMAGER SYSTEM (imaging system) (Alpha Innotech, San Leandro, Calif. USA) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. The assays were all performed in duplicate for each peptide antigen.
(36) Results
(37) Binding of Survivin Derived Peptides to HLA A2
(38) The amino acid sequence of the survivin protein was screened for the most probable HLA-A2 nona- and decamer peptide epitopes, using the main HLA-A2 specific anchor residues (17). Ten survivin-derived peptides were synthesized and examined for binding to HLA-A2. An epitope from HIV-1 pol476-484 (ILKEPVHGV, SEQ ID NO:11) (Table 1) was used as a positive control. The peptide concentration required for half maximal recovering of class I MHC (C.sub.50 value) was 0.7 M for the positive control. In comparison, the peptide designated Sur9 (ELTLGEFLKL, SEQ ID NO:3) bound at an affinity of C.sub.50=10 M. The peptides designated Sur6 (FLKLDRERA, SEQ ID NO:1) and Sur8 (TLPPAWQPFL, SEQ ID NO:2), respectively bound to HLA-A2 at C.sub.50=30 M, whereas Sur1 (LTLGEFLKL, SEQ ID NO:10) and Sur3 (KVRRAIEQL, SEQ ID NO:13) bound weaker (C.sub.50>100 M). Five of the peptides examined (Sur2, Sur4, Sur5, Sur7, and Sur10) did not bind to HLA-A2.
(39) Since Sur1 is a weak HLA-A2 binder, two analogue peptides designated Sur1L2 and Sur1M2, respectively in which a better anchor residue (leucine or methionine) replaced the native threonine at position 2 were synthesized. Both of these peptides bind with almost similar high affinity to HLA-A2 as the positive control (C.sub.50=1 M).
(40) CTL Response to Survivin in CLL Patients
(41) PBLs from four HLA-A2 positive CLL patients were stimulated once in vitro before examination in the ELISPOT assay. This procedure was chosen to extend the sensitivity of the ELISPOT. All of the above 10 survivin-derived peptides were included in the first line of experiments. Responses were detected to Sur1 and Sur9 and only data for these peptides are given in the figures.
(42) CTL Response to Survivin in Melanoma Patients
(43) T lymphocytes isolated from tumour infiltrated lymph nodes from HLA-A2 positive melanoma patients were examined. The freshly resected lymph node was minced into small fragments and crushed to release cells into culture. Cells were stimulated once with peptide in vitro before examination in the ELISPOT assay. Survivin specific T cells were detested in three of the six patients analyzed. A strong Sur9 response was detected in patient Mel2 and Mel3. A weaker response to the Sur1 peptide was also detected in these patients (
Example 2
Spontaneous Cytotoxic T-Cell Responses to Survivin-Derived MHC Class I-Restricted T-Cell Epitopes In Situ and Ex Vivo in Cancer Patients
(44) Summary
(45) Spontaneous cytotoxic T-cell responses to survivin-derived MHC class I restricted T-cell epitopes were demonstrated in situ as well as ex vivo in breast cancer, leukemia, and melanoma patients. Moreover, survivin reactive T cells isolated by magnetic beads coated with MHC/peptide complexes were cytotoxic to HLA-matched tumours of different tissue types. Being a universal tumor antigen, survivin may serve as a widely applicable target for anti-cancer immunotherapy.
(46) Materials and Methods
(47) Construction of HLA-Peptide Complexes for T-Cell Staining and T-Cell Sorting
(48) A recognition site for enzymatic biotinylation using biotin protein ligase (BirA) in fusion with the 5-end of the extracellular domains of HLA A*0201 (residues 1-275) was expressed in E. coli BL21 (DE3). The recombinant protein was purified by size (SEPHADEX G 25 (gel filtration), Pharmacia) and ion exchange (MONO-Q, Pharmacia) chromatography from inclusion bodies solubilised in 8 M urea. The HLA A*0201 was folded in vitro by dilution in the presence of the modified survivin peptide Sur1M2 (LMLGEFLKL, SEQ ID NO:5) or the MAA peptide gp100154-163, and subsequently biotinylated as described previously (35, 36).
(49) After gel filtration on a Pharmacia SEPHADEX G 25 (gel filtration) column to remove unbound biotin, the protein was multimerised with streptavidin-FITC conjugated dextran molecules (kindly provided by L. Winther, DAKO, Denmark) to generate multivalent HLA-dextran compounds for immunohistochemistry. The HLA A*0201 construct was a kind gift of Dr. Mark M. Davis (Dept. of Microbiology and Immunology, Stanford University, Palo Alto, Calif.). Cell separation was performed as previously described (37). Briefly, 5106 streptavidin-conjugated magnetic beads (Dynal, Oslo, Norway) were washed twice in 200 l cold PBS, 0.5 g peptide/A*0201 monomers were added and the mixture incubated for 15 min. at room temperature. After two washes these beads were mixed with PBLs at a ratio of 1:10 and subsequently incubated for 1 h followed by a precipitation of bead-bound cells in a magnetic field. The precipitation step was repeated once.
(50) Immunohistochemistry Stainings
(51) For staining with FITC-conjugated multimeric peptide/MHC complexes, tissue sections were dried overnight and subsequently fixed in cold acetone for 5 min. All incubation steps were performed at room temperature and in the dark: (i) 45 min. of the primary antibody (1:100 diluted), (ii) Cy 3-conjugated goat anti-mouse (1:500 diluted; code 115-165-100, Jackson ImmunoResearch, obtained from Dianova, Hamburg, Germany) for 45 min. and finally (iii) the multimers for 75 min. Between each step the slides were washed two times for 10 min. in PBS/BSA 0.1%. The slides were mounted in VECTASHIELD (antifade mounting medium) and kept in the refrigerator until observed under the confocal microscope.
(52) Cytotoxicity Assay
(53) Conventional [51Cr]-release assays for CTL-mediated cytotoxicity were carried out as described in (13). Target cells were autologous EBV-transformed B-cell lines, the HLA-A2 positive breast cancer cell line MCF-7 (available at ATCC), the HLA-A2 positive melanoma cell line FM3 (38), the HLA-A2 negative breast cancer cell line BT-20 (available from ATCC) and the HLA-A2 negative melanoma cell line FM45 (38). All cancer cell lines expressed survivin as examined by RT-PCR (data not shown).
(54) ELISPOT Assay
(55) The ELISPOT assay was used to quantify peptide epitope-specific IFN- releasing effector cells and has been described previously (39). Briefly, nitrocellulose bottomed 96-well plates (MULTISCREEN (96-well assay plate) MAIP N45, Millipore) were coated with an anti-IFN- antibody (1-D1K, Mabtech, Sweden) and non-specific binding was blocked using AIM V medium (serum-free media) (GibcoBRL, Life Technologies Inc., Gaithersburg, Md., USA). Lymphocytes were added at different cell concentrations together with the specific peptides and T2 cells and incubated overnight at 37 C. Following two washes the biotinylated detection antibody (7-B6-1-Biotin, Mabtech) was added. Specific binding was visualised using alkaline phosphatase-avidin together with the respective substrate (GibcoBRL). The reaction was terminated upon the appearance of dark purple spots, which were quantitated using the ALPHA IMAGER SYSTEM (imaging system) (Alpha Innotech, San Leandro, Calif., USA). The peptides used for the ELISPOT were Sur1, Sur9 and the Sur1 analogue peptide Sur1M2 as described in Example 1.
(56) Results
(57) In Situ Staining of HLA-A2/Survivin Reactive T Cells
(58) In Example 1 two survivin-derived peptide epitopes recognized by T cells in leukemia and melanoma, i.e., Sur1 were identified. The weak binding affinity of Sur1 to HLA-A2 was improved substantially by replacing threonine at position 2 with a better anchor residue (methionine; Sur1M2). This measure enabled the construction of stable HLA-A2/peptide complexes. These complexes were multimerised using dextran molecules, which were conjugated with streptavidin and FITC. Multimerised MHC-complexes were used to stain acetone-fixed, frozen material. Using a confocal laser microscope, Sur1M2/HLA-A*0201 reactive CTLs could readily be detected in situ in the tumor microenvironment. We depicted such cells in the primary tumor and the sentinel lymph node of a stage III melanoma patient as well as in a primary breast cancer lesion. To ensure the specificity of the staining, a series of negative controls was carried out. Neither the use of peptide/HLA-dextran multimers with peptides derived from the melanoma differentiation antigen gp100 on the same tumour, nor Sur1M2/HLA-dextran multimers in case of a tumour sample obtained from an HLA-A2 negative donor resulted in a positive staining.
(59) Isolated Survivin Reactive CTLs Lyse Tumour Cell Lines of Different Origin
(60) To characterise the functional capacity of survivin-reactive CTLs, these cells were isolated by means of magnetic beads coated with HLA-A2/Sur1M2-complexes (36). A freshly resected melanoma infiltrated lymph node was minced into small fragments and crushed to release cells Into culture. Cells were stimulated once with peptide in vitro prior to isolation. One day after isolation IL-2 was added, and on day 5 the capacity of these cells to kill tumour cells was tested either by ELISPOT or in standard 51Cr release assays. First, by means of ELISPOT analysis it was possible to establish that CTLs isolated using the modified Sur1M2/HLA-A2-complex also responded to the native Sur1 peptide (data not shown). Second, the cytotoxicity of the survivin reactive CTLs against the HLA-A2 positive melanoma cell-line FM3 (
(61) Survivin Reactivity Measured in PBL by ELISPOT
(62) The presence of survivin reactive T cells in PBLs from ten HLA-A2 positive breast cancer patients was examined by the ELISPOT. Before analysis, PBLs were stimulated once in vitro to extend the sensitivity of the assay. Reactivity to the following survivin peptides was examined: Sur1, Sur9 and Sur1M2. Survivin specific T cells were detected in six out of the ten HLA-A2 positive breast cancer patients. Representative examples are given in
(63) TABLE-US-00004 TABLE 2 Patients with survivin peptide-specific T lymphocytes in PBLs as measured by ELISPOT Patient Sur1 Sur9 Sur1M2 Melanoma a) P4 97 P11 112 P13 71 P15 61 101 P17 172 P39 127 P64 112 70 128 Breast cancer b) B1 122 208 B2 67 72 B3 54 B4 45 B5 19 B6 24 CLL c) CLL1 27 320 CLL2 39 CLL3 23 127 122 CLL5 100 124 CLL6 121 360 CLL7 68 132 174 a) Frequency of reactive cells per 10.sup.4; 14 patients examined. b) Frequency of reactive cells per 10.sup.4; 10 patients examined. c) Frequency of reactive cells per 10.sup.5; 7 patients examined.
Example 3
HLA-B35-Restricted Immune Responses to Survivin-Derived Peptides in Cancer Patients
(64) Summary
(65) In this study, two survivin-derived epitopes, which are restricted to HLA-B35 were identified and characterized. Specific T-cell reactivity against both of these epitopes was present in the peripheral blood from patients with different haematopoietic malignancies and melanoma. Substitutions of the C-terminal anchor residue improved the recognition by tumor infiltrating lymphocytes from melanoma patients. Furthermore, spontaneous cytotoxic T-cell responses to survivin in situ in a primary melanoma lesion was demonstrated. These epitopes extends the applicability of future vaccine strategies based on survivin peptides in relation to malignancies as well as the HLA profile of the patients involved.
(66) In Examples 1 and 2, HLA-A2 restricted survivin-derived T-cell epitopes were studied. Since HLA-A2 is only expressed in about 30% of the Caucasian population (63), peptide epitopes restricted to other HLA class I molecules need to be identified to extend the fraction of patients that could be treated. In this study, two novel T-cell epitopes from survivin restricted to HLA-B35, which is expressed in 9% of the Caucasian population (63), were identified, and spontaneous immune responses to these survivin peptides were detected in patients with different haematopoietic malignancies and melanoma.
(67) Materials and Methods
(68) Patients
(69) Peripheral vein blood samples from cancer patients were collected, PBLs were isolated using LYMPHOPREP (density gradient medium) separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen) and frozen in FCS with 10% DMSO. Ten HLA-B35 positive patients were selected for further analysis. These patients suffered from melanoma, CLL, follicular lymphoma (FL), diffuse large B-cell lymphomas (DLBCL) and Multiple Myeloma (MM), respectively. At the time blood samples were collected patients had not been medically treated within the previous four months. Additionally, tumor-infiltrating lymphocytes (TIL) isolated from lymph nodes were collected from three of the melanoma patients and frozen in FCS with 10% DMSO.
(70) Peptides
(71) Seven synthetic survivin-derived peptides were used in this study: Sur6-14, Sur11-19, Sur34-43, Sur46-54, Sur51-59, Sur46Y9, Sur51Y9, and one EBV-derived peptide, EBNA3A 457-466 (63). All peptides were obtained from Research Genetics (Huntsville, Ala.) and provided at >90% purity, as verified by HPLC and MC analyses. The peptides are listed in Table 3 below.
(72) TABLE-US-00005 TABLE3 HLA-B35bindingofsurvivin-derivedpeptides Name Proteinandposition Sequence SEQIDNO: C.sub.50(M) Sur6-14 Survivin.sub.6-14 LPPAWQPFL 18 >100 Sur11-19 Survivin.sub.11-19 QPFLKDHRI 19 Notbinding Sur34-43 Survivin.sub.34-43 TPERMAEAGF 20 >100 Sur46-54 Survivin.sub.46-54 CPTENEPDL 6 20 Sur51-59 Survivin.sub.51-59 EPDLAQCFF 7 13 Sur46Y9 Modifiedpeptide CPTENEPDY 8 4 Sur51Y9 Modifiedpeptide EPDLAQCFY 9 1.5 C24 EBNA3A.sub.458-466 YPLHEQHQM 21 0.8
Assembly Assay for Peptide Binding to MHC Class I Molecules
(73) The assembly assay described in Examples 1 and 2 was used to measure binding affinity of the synthetic peptides to HLA-B35 molecules metabolically labeled with [S35]methionine. Briefly, the assay is based on peptide-mediated stabilization of empty HLA molecules released, upon cell lysis, from the TAP deficient cell line T2, stably transfected with HLA-B35 (kindly provided by Dr J. Haurum, Symphogen ApS, Lyngby, Denmark). Stably folded HLA-molecules were immunoprecipitated using the conformation-dependent mAb W6/32. The HLA molecules were separated by IEF electrophoresis, gels were exposed to phosphorimager screens (Imaging plate, FUJI photo film Co., LTD., Japan), analyzed and the amount of correctly folded HLA molecules were quantified using ImageGauge phosphorimager software (FUJI photo film Co., LTD., Japan).
(74) Antigen Stimulation of PBLs
(75) To extend the sensitivity of the ELISPOT assay, lymphocytes were stimulated once in vitro with peptide prior to analysis (14, 15). PBLs or TILs were thawed and stimulated with 50 M of the individual peptide epitopes in 96-well plates for 2 h at 26 C. (510.sup.5-10.sup.6 cells per peptide), and pooled for further 10 days of culture at 37 C. in x-vivo with 5% human serum (HS), in 24 well plates (Nunc, Roskilde, Denmark), with 210.sup.6 cells per well. At the second day of incubation 40 g/ml IL-2 (Apodan A/S, Denmark) were added. At day 10, the cultured cells were tested for reactivity in the ELISPOT assay.
(76) The ELISPOT Assay
(77) The ELISPOT assay used to quantify peptide specific, IFN- releasing effector cells in PBLs or TILs collected from cancer patients was performed as described in Example 1. Briefly, nitrocellulose-bottomed 96-well plates (MULTISCREEN (96-well assay plate) MAIP N45; Millipore, Hedehusene, Denmark) were coated with mAb against human IFN-, 7.5 g/ml (1-D1K; Mabtech, Nacka, Sweden). Wells were washed and blocked in x-vivo (X-VIVO 15 BioWhittacker (chemically defined, serum-free media), Molecular Applications Aps, Denmark) and cells were added in duplicates at different concentrations. For antigen presentation, 10.sup.4 T2-B35 cells, with and without 10 M peptide, were added per well. Plates were incubated overnight, the cells discarded, and wells washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin; Mabtech). Plates were incubated 2 h at room temperature, washed and avidin-alkaline phosphatase conjugate was added (AP-Avidin; Calbiochem, Life Technologies, Inc.). After 1 h of incubation at room temperature, the enzyme substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Code No. K0598, DakoCytomation Norden A/S) was added, and dark purple spots emerged in 3-7 min. The reaction was terminated by washing with tap water. Spots were counted using the ALPHA IMAGER SYSTEM (imaging system) (Alpha Innotech, San Leandro, Calif.), and the frequency of peptide specific T cells were calculated from the number of spot forming cells.
(78) All assays were performed in duplicates for each peptide antigen, and lymphocytes cultured in the same well, were tested in equal cell numbers with and without peptide, to measure the number of peptide specific cells in the culture.
(79) Maturation of Dendritic Cells (DCs)
(80) Adherent cells were isolated from PBLs after 2 h of culture. These were cultured for 10 additional days in RPMI 1640 (GIBCO, Invitrogen Corporation, UK) with 10% FCS. 800 ng/ml GM-CSF (PreproTech, London, UK) and 40 ng/ml IL-4 (PreproTech) were added every third day. At day 10, DCs were matured for 24 h by adding 50 ng/ml TNF-.alpha. (PreproTech). After maturation, DCs were released and pulsed with 20 M peptide in the presence of 3 g/ml. 2-microglobulin for 2 h at 26 C.
(81) Isolation of Peptide Specific T Cells
(82) Antigen specific cells were isolated using sur51Y9/HLA-B35-coated magnetic beads as described in Example 2. Biotinylated monomers of HLA-B35 with sur51Y9 (obtained from ProImmune, Oxford, UK) were coupled to streptavidin coated magnetic beads (DYNABEADS M-280 (magnetic beads), Dynal A/S, Oslo, Norway) by incubating 2.5 g monomers with 510.sup.6 beads in 40 l PBS for 20 min. at room temperature. The magnetic complexes were washed three times in PBS, using a magnetic device (Dynal A/S, Oslo, Norway) and subsequently mixed with PBLs at a ratio of 1:10 in PBS with 5% BSA, and rotated very gently for 1 h. Antigen specific CD8+ T cells associating with the magnetic complexes were gently washed two or three times. Isolated cells were resuspended several times in x-vivo supplemented with 5% human serum and incubated for 2 h before the magnetic beads were released and removed from the cell suspension. The isolated antigen specific CD8+ T cells were used in ELISPOT assay to analyze the cross-reactivity between the native and modified peptide.
(83) TCR Clonotype Mapping by Denaturing Gradient Gel Electrophoresis (DGGE)
(84) DGGE clonotype mapping of the human TCR BV regions 1-24 has been described in details (66). Briefly, RNA was isolated using the PURESCRIPT Isolation Kit (RNA isolation kit) (Gentra Systems Inc. MN) and transcribed cDNA was amplified by PCR using primers for the variable regions of the TCR beta chains in conjunction with a common constant region primer. The computer program MELT87 was used to ensure that the amplified DNA molecules were suited for DGGE analysis provided a 50 bp GC-rich sequence (GC-clamp) was attached to the 5-end of the constant region primer. DGGE analysis was done in 6% polyacrylamide gels containing a gradient of urea and formamide from 20% to 80%. Electrophoresis was performed at 160 V for 4.5 hours in 1TAE buffer at a constant temperature of 54 C.
(85) Immunohistochemistry Stainings
(86) Multimerised peptide/HLA complexes were used to identify antigen specific T cells in situ in tumor lesions of cancer patients using the procedure described in Example 2. Biotinylated sur51Y9/HLA-B35 monomer was supplied by Proimmune limited, Oxford, UK. The biotinylated monomers of sur51Y9/HLA-B35 were multimerised with streptavidin-FITC-conjugated dextran molecules (kindly provided by L. Winther, DAKO, Glostrup, Denmark) to generate multivalent HLA-dextran compounds for immunohistochemistry. Tissue sections were dried overnight and subsequently fixed in cold acetone for 5 min. All the incubation steps were performed in the dark at room temperature: (a) 45 min of the primary antibody (1:100 diluted) (b) Cy 3-conjugated goat-anti-mouse antibody (1:500 diluted; code 115-165-100; Jackson ImmunoResearch, obtained from Dianova, Hamburg, Germany) for 45 min; and finally (c) the multimers for 75 min. Between each step, the slides were washed two times for 10 min in PBS/BSA 0.1%. The slides were mounted in VECTASHIELD (antifade mounting medium) and kept in the refrigerator until observed under the confocal microscope (Leica).
(87) Results
(88) Identification of HLA-835 Binding Survivin-Derived Peptides
(89) The amino acid sequence of survivin was screened for nonameric and decameric peptides with anchor residues, according to the peptide-binding motif of HLA-B35 (67). Five peptides were selected containing proline as the N-terminal anchor in position 2 and phenylalanine, leucine, isoleucine or tyrosine as C-terminal anchor residues (Table 3). Assembly assay revealed two peptides, sur51-59 (EPDLAQCFF, SEQ ID NO:7) and sur46-54 (CPTENEPDL, SEQ ID NO:6) that were able to stabilise HLA-B35 efficiently. Additionally, two peptides, sur34-43 (TPERMAEAGF, SEQ ID NO:20) and sur6-14 (LPPAWQPFL, SEQ ID NO:18) showed a weak stabilization, whereas the remaining peptide did not stabilize HLA-B35 at all. The peptide concentration required for half maximal recovery of HLA-B35 (C50) was estimated at 13 M for sur51-59 and 20 M for sur46-54. In comparison, the positive control-epitope C24 from EBNA3A458-466 (YPLHEQHQM, SEQ ID NO:21) had an estimated C.sub.50 value of 0.8 M.
(90) To enhance the binding affinity of sur46-54 and sur51-59 the C-terminal amino acid was replaced with tyrosine, a better anchor residue (67). The recovery of HLA-B35 mediated by the modified peptides was analyzed in the assembly assay, and C.sub.50 values were estimated at 1.5 M for sur51Y9 and 4 M for sur46Y9 (
(91) Spontaneous Immune Responses Against Native Peptide Epitopes
(92) Initially, five patients were analyzed for spontaneous immune responses to the four native HLA-B35 binding peptides sur51-59, sur46-54, sur34-43 and sur6-14. These five patients had different haematopoietic malignancies: HEM8 and HEM18 suffered from MM, HEM12 from FL, HEM9 had DLBCL, and CLL5 had CLL.
(93) INF- ELISPOT assays were performed on PBLs after 10 days of in vitro stimulation to detect peptide precursor CTLs. Spontaneous immune responses were detected against two of the native HLA-B35 binding peptides, sur51-59 and sur46-54. Two patients, HEM12 and CLL5 showed a response to both sur51-59 and sur46-54, whereas HEM8 only showed a response to sur51-59 (
(94) An alternative approach to in vitro stimulation was used in patient HEM12, i.e. PBLs were co-cultured with matured autologous dendritic cells pulsed with sur51-59 to stimulate a CTL response in vitro. PBLs from this culture showed strong reactivity towards sur51-59 in ELISPOT (
(95) Increased Recognition of Modified Peptides
(96) As described above, peptide modifications to enhance the HLA-B35 affinity resulted in a 5-10-fold higher affinity for HLA-B35 relative to the native peptides. A group of five melanoma patients were analyzed for spontaneous immune responses to both the native and modified peptides by means of ELISPOT assay. PBL samples were analyzed after in vitro stimulation, whereas TIL samples were analyzed directly. Spontaneous immune responses were observed in either PBLs or TILs from three of the five patients. FM25 showed reactivity against sur51-59 and sur51Y9 in both PBL and TIL samples (
(97) Cross-Reactivity Between the Native and Modified Peptide
(98) The high affinity of sur51Y9 to HLA-B35 enables the production of stable monomers of HLA-B35 with sur51Y9. Having established the presence of survivin reactive T lymphocytes in tumor infiltrated lymph nodes and PBLs from different cancer patients, magnetic beads were coated with such HLA-B35/Sur51Y9-complexes and these were used to isolate survivin peptide reactive T lymphocytes from PBL from patient CLL5. This patient showed a strong response to sur51-59. Beads were tightly bound to the cell surface of the specific cells, as visualized by microscopy (data not shown), permitting precipitation of antigen specific cells by a magnetic field. The isolated sur51Y9 specific cells responded strongly to sur51-59, (
(99) Antigen Specific T Cells Present In Situ in a Melanoma Lesions
(100) Sur51Y9/HLA-B35 monomers were multimerised using dextran molecules conjugated with streptavidin and FITC. Multimerised MHC-complexes were used to stain acetone-fixed, frozen material using the procedure described in Example 2. Antigen specific cells were visualized using a confocal laser microscope. Sections of primary melanoma from three patients were analyzed, and Sur51Y9/HLA-B35-reactive CTLs could readily be detected in situ in the tumor microenvironment in one of the patients. Co-staining with a mAb against granzyme B showed that these survivin specific CTLs released granzyme B, exerting cytotoxic activity, HLA-B35 negative melanoma patients were used as controls (data not shown).
Example 4
Identification of Novel Survivin-Derived CTL Epitopes with Different HLA-A-Restriction Profiles
(101) Summary
(102) Novel HLA-A1-, HLA-A2-, HLA-A3- and HLA-A11-restricted survivin epitopes were characterised on the basis of CTL responses in cancer patients. These epitopes significantly increase the number of patients eligible for immunotherapy based on survivin-derived peptides. Additionally, the collective targeting of several restriction elements is likely to decrease the risk of immune escape by HLA-allele loss.
(103) Materials and Methods
(104) Patients
(105) Patient samples were received from the University of Wrzburg, Germany and the University Hospital in Herlev, Denmark. Informed consent was obtained from the patients prior to any of these measures. Tissue typing was conducted at Department of Clinical Immunology, University Hospital, Copenhagen, Denmark. Peripheral blood lymphocytes (PBL) from cancer patients with melanoma, mamma carcinoma, and chronic lymphocytic leukemia (CLL) were isolated using Lymphoprep separation and frozen in fetal calf serum (FCS) with 10% dimethylsulphoxide. Furthermore, T lymphocytes from primary lesions and from tumor infiltrated lymph nodes from melanoma patients were obtained. Freshly resected tumor tissue was minced into small fragments, and crushed to release tumor-infiltrating lymphocytes (TIL) for cryopreservation.
(106) Peptides
(107) All peptides were purchased from Invitrogen (Carlsbad, Calif., USA) and provided at >80% purity as verified by HPLC and MS analysis. All peptides used are listed in Table 4, Example below.
(108) Cell Lines
(109) The human T2 cell line is a TAP1 and TAP2 defective hybrid of the B-LCL.174 and the T-LCL CEM cells and thus only express low levels of HLA class I molecules (HLA-A*0201 and HLA-B*5101) at the cell surface. T2 cells transfected with HLA-A*0301 were kindly provided by Dr A McMicheael, IMM, John Radcliffe Hospital, Oxford. T2 cells transfected with HLA-A*1101 were kindly provided by Dr M Masucci, MTC, Karolinska Institute, Stockholm, Sweden. The BM36.1 cell line is also defective in TAP function and has a similar phenotype as T2 with low expression of HLA class I (HLA-A*0101, HLA-B*3501) at the surface. The BM36.1 cells were kindly provided by Dr A Ziegler, Humboldt University, Berlin, Germany.
(110) Assembly Assay for Peptide Binding to MHC Class I Molecules
(111) The binding affinity of synthetic peptides (Invitrogen, Carlsbad, Calif., USA) to HLA-A1, -A2, -A3, or -A11 molecules metabolically labeled with [.sup.35S]-methionine was measured in the assembly assay, as described previously (12). The assay is based on peptide-mediated stabilization of empty HLA molecules released upon cell lysis, from the TAP-deficient cell lines. Stably folded HLA-molecules were immune-precipitated using the HLA class I-specific, conformation-dependent mAb W6/32, and separated by isoelectric focusing (IEF) gel electrophoresis. MHC heavy chain bands were quantified using the ImageGauge Phosphorimager program (FUJI photo film Co., Carrollton, Tex., USA). The intensity of the band is proportional to the amount of peptide-bound class I MHC complex recovered during the assay. Subsequently, the extent of stabilization of the HLA-molecule is directly related to the binding affinity of the added peptide. The peptide concentration used to analyze the recovery of the HLA-molecules was 40, 4, 0.4, 0.04 M for HLA-A1 and HLA-A11, and 100, 10, 1, 0.1, 0.01 M for HLA-A2 and HLA-A3. The C.sub.50 value was subsequently calculated for each peptide as the peptide concentration sufficient for half maximal stabilization.
(112) Antigen Stimulation of PBL
(113) To extend the sensitivity of the ELISPOT assay, PBL were stimulated once in vitro prior to analysis. At day 0, PBL or crushed lymph nodes were thawed and plated as 210.sup.6 cells in 2 ml/well in 24-well plates (Nunc, Roskilde, Denmark) in x-vivo medium (Bio Whittaker, Walkersville, Md.), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 M of peptide. Two days later 20 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 10.
(114) ELISPOT Assay
(115) The ELISPOT assay was used to quantify peptide epitope-specific interferon-.gamma. releasing effector cells as previously described (16). Briefly, nitrocellulose bottomed 96-well plates (MULTISCREEN MAIP N45 (96-well assay plate), Millipore, Hedehusene, Denmark) were coated with anti-IFN- antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo medium, and the cells were added in duplicates at different cell concentrations. The peptides were then added to each well and the plates were incubated overnight. The following day, media was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed, and avidin-alkaline phosphatase conjugate (Calbiochem, Life Technologies, Inc. San Diego, Calif., USA) was added to each well. The plates were incubated at room temperature for one hour, washed, and the enzyme substrate NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate) (DakoCytomation Norden A/S, Glostrup, Denmark) was added to each well and incubated at RT for 5-10 min. Upon the emergence of dark purple spots, the reaction was terminated by washing with tap-water. The spots were counted using the IMMUNOSPOT Series 2.0 Analyzer (plate reader) (CTL Analyzers, LLC, Cleveland, US) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. All assays were performed in duplicates for each peptide antigen.
(116) Results
(117) Identification of HLA-A1 Restricted Survivin Epitopes
(118) Binding of Survivin-Derived Peptides to HLA A1
(119) The amino acid sequence of the survivin protein was screened for the most probable HLA-A1 nonamer or deca-mer peptide epitopes, using the main HLA-A1 anchor residues, aspartic acid (D), glutamic acid (E) at position 3 and tyrosine (Y), phenylalanine (F) at the C-terminus. Accordingly, six survivin-derived peptides were synthesized and examined for binding to HLA-A1 (table 4). Additionally, the two peptides Sur38-46 (MAEAGF1HC)(SEQ ID NO:23) and Sur47-56 (PTENEPDLAQ) (SEQ ID NO:25) was included, in spite they only contain one of the main anchors, since both were identified as possible good binders by the predictive algorithm by Rammensee et al. C.sub.50 values were estimated for each peptide as the peptide concentration needed for half maximal stabilization of HLA-A1 (table 4). However, only one of these peptides Sur92-101 (QFEELTLGEF) (SEQ ID NO: 27) bound with almost similar high affinity as a known positive control epitope from the Influenza A protein, basic polymerase 1 (PB1) (VSDGGPNLY) as exemplified in
(120) HLA-A1 Restricted CTL Responses Against Survivin-Derived Peptides in Cancer Patients
(121) PBL from six melanoma patients and TIL from three melanoma patients were analyzed for the presence of CTL specific against any of the four high affinity survivin deduced peptides Sur38Y9, Sur47Y10, Sur92-101, and Sur93T2 by means of ELISPOT. T-cell reactivity against at least one of the survivin-derived peptides was observed in three PBL samples and one TIL sample from the total of nine patients analyzed. As seen in
(122) In addition, ten melanoma patients were tested for immune reactivity against the native peptides Sur93-101, Sur38-46 and Sur47-56 by means of ELISPOT; however, no peptide-specific responses were detected in any of these patients (data not shown).
(123) Identification of HLA-A11 Restricted Survivin Epitopes
(124) Binding of Survivin-Derived Peptides to HLA-A11
(125) The amino acid sequence of the survivin protein was screened for nonamer or deca-mer peptides with binding motifs corresponding to that of the HLA-A3 super-family, including HLA-A3 and HLA-A11. Peptide sequences with the main anchor residues, leucine (L) in position 2 and lysine (K) at the C-terminus, were chosen together with peptide sequences having related amino acids at these positions according to the predictive algorithm by Rammensee et al. (table 4).
(126) Thirteen peptides were predicted from the protein sequence of survivin and analyzed for binding to HLA-A11 and HLA-A3. Three of these peptides, Sur53-62 (DLAQCFFCFK) (SEQ ID NO:47), Sur54-62 (LAQCFFCFK) (SEQ ID NO:42) and Sur112-120 (KIAKETNNK) (SEQ ID NO:44) bound HLA-A11 with high affinity, comparable to the viral epitope from EBV nuclear antigen 4 (AVFDRKSDAK) (SEQ ID NO:63). In addition, one peptide, Sur112-121 (KIAKETNNKK) (SEQ ID NO:51) bound weakly to HLA-A11 (Table 4).
(127) HLA-A11 Restricted CTL Responses Against Survivin-Derived Peptides in Cancer Patients
(128) PBL from five melanoma patients and two CLL patients were tested for T-cell reactivity against the four HLA-A11 binding peptides Sur53-62; Sur54-62, Sur112-120, and Sur112-121. We were able to detect responses against the survivin-derived peptide Sur53-62 in PBL from two of the melanoma patients, Mel.A11-1, Mel.A11-2, by means of ELISPOT (
(129) Identification of HLA-A3 Restricted Survivin Epitopes
(130) Binding of Survivin-Derived Peptides to HLA-A3
(131) The survivin-derived peptides predicted for binding to the HLA-A3 super-family were additionally analyzed for the binding to HLA-A3. Only two of the peptides Sur112-120 (KIAKETNNK) (SEQ ID NO:44) and Sur112-121 (KIAKETNNKK) (SEQ ID NO:57) bound HLA-A3 with high affinity, similar to the viral epitope, Influenza A nucleoprotein 265-273 (ILRGSVAHK) (SEQ ID NO:74) (Table 4). Furthermore, two peptides Sur53-62 (DLAQCFFCFK) (SEQ ID NO:47) and Sur95-103 (ELTLGEFLK) (SEQ ID NO:43) bound weakly to HLA-A3.
(132) Some of the peptides with no detectable binding were modified in an attempt to increase the binding affinity for HLA-A3. Thus, we synthesized two analogue peptides of Sur54-62 and Sur113-122 in which a better anchor residue leucine (L) replaced the natural alanine (A) at position 2. Sur54L2 (LLQCFFCFK) (SEQ ID NO:56) bound HLA-A3 with high affinity, whereas Sur113L2 (ILKETNNKKK) (SEQ ID NO:59) only bound weakly (Table 4). In addition, we synthesized four analogue peptides of Sur5-13, Sur13-22, Sur18-27, and Sur53-61 in which the better anchor residue lysine (K) replaced the natural phenylalanine (F) at the C-terminus. Sur5K9 (TLPPAWQPK) (SEQ ID NO:54) and Sur18K10 (RISTFKNWPK) (SEQ ID NO:58) bound to HLA-A3 with high affinity, whereas the substitutions had no detectable effect on the binding to HLA-A3 of Sur13K9 (FLKDHRISTK) (SEQ ID NO:57) and Sur53K9 (DLAQCFFCK) (SEQ ID NO:55) compared to the native analogues.
(133) HLA-A3 Restricted CTL Responses Against Survivin-Derived Peptides in Cancer Patients
(134) Nine samples from melanoma patients (five PBL and four TIL) were analyzed for immune reactivity against the two native high affinity HLA-A3 binding peptides Sur112-120 and Sur112-121, as well as the two native, weak binding peptides Sur53-62 and Sur95-103. However, no immune responses against these peptides could be detected by ELISPOT in any of the patients. Subsequently, the same patients were analyzed for spontaneous immune reactivity against the three high affinity, modified survivin-derived peptides, Sur5K9, Sur18K10, and Sur54L2. CTL reactivity was detected against Sur18K10 in TIL samples from three patients, Mel.A3-1, Mel.A3-2, Mel.A3-3 (
(135) Identification of a Novel HLA-A2 Restricted Survivin Epitope
(136) Binding of 11-Mer Survivin-Derived Peptides to HLA-A2
(137) The amino acid sequence of the survivin protein was screened for the most probable HLA-A2 11-mer peptide epitopes, using the main HLA-A2 specific anchor residues. Six survivin deduced peptides were synthesized and examined for binding to HLA-A2. None of the peptides examined bound with similar high affinity as a known positive control epitope from Epstein-Barr virus BMLF.sub.280-288 peptide (GLCTLVAML) (SEQ ID NO:72) (Table 4). The peptide concentration required for half maximal recovery of HLA-A2 (C.sub.50 value) was 0.9 M for the positive control. In comparison, the peptides Sur18-28 (RISTFKNWPFL) (SEQ ID NO:67) and Sur86-96 (FLSVKKQFEEL) (SEQ ID NO:69) bound weakly to HLA-A2 (C.sub.50=69 M and 72 M respectively). However, the two known HLA-A2-restricted survivin epitopes bound in a similar way weakly to HLA-A2; Sur95-104 (ELTLGEFLKL) (SEQ ID NO:43) bound with intermediate affinity (C.sub.50=10 M) whereas Sur96-104 (LTLGEFLKL) (SEQ ID NO:10) bound only weakly (C.sub.50>100 M). The remaining four 11-mer peptides examined (Sur-4-14 (PTLPPAWQPFL) (SEQ ID NO:66), Sur54-64 (LAQCFFCFKEL) (SEQ ID NO:68), Sur88-98 (SVKKQFEELTL) (SEQ ID NO:70), and Sur103-113 (KLDRERAKNKI) (SEQ ID NO:74)) did not bind to HLA-A2.
(138) HLA-A2 Restricted CTL Responses Against Survivin-Derived Peptides in Cancer Patients
(139) PBL from ten cancer patients (two melanoma (Mel), six CLL (CLL), and two mamma carcinoma (MC) patients) was initially analyzed to investigate whether the two weak binding 11mer peptides, Sur18-28 and Sur86-96 were presented by HLA-A2 and recognized by the immune system of cancer patients. CTL responses against Sur18-28 were found in PBL from two of the ten patients analyzed (CLL-1, CLL-2,
(140) Identification of HLA-B7 Restricted Survivin Epitopes
(141) Binding of Survivin Derived Peptides to HLA-B7
(142) The amino acid sequence of the survivin protein was screened for peptides of nine to ten amino acids, with anchor residues according to the peptide binding motif of HLA-B7. Five peptides were selected and analyzed for their ability to stabilize HLA-B7 in the assembly assay. C.sub.50 values were estimated for each peptide as the peptide concentration needed for half maximal stabilization of HLA-B7 (table 4). Two survivin-derived peptides, sur6-14 (LPPAWQPFL) (SEQ ID NO:18) and sur11-19 (QPFLKDHRI) (SEQ ID NO:19) stabilized HLA-B7 weakly, with C.sub.50 values above 100 M; whereas sur46-54 (CPTENEPDL) (SEQ ID NO:6), sur51-59 (EPDLAQCFF) (SEQ ID NO:7), and sur34-43 (TPERMAEAGF) (SEQ ID NO:20) did not bind to HLA-B7 (table 4).
(143) HLA-B7 Restricted CTL Responses Against Survivin Derived Peptides in Cancer Patients
(144) HLA-B7 positive PBL from five melanoma patients (mel25, mel26, mel3, mel6, mel39), two CLL patients (CLL1, CLL54) and 2 breast cancer patients (breast11, breast 15) were tested for T-cell reactivity against the weak HLA-B7 binding peptides sur6-14 (LPPAWQPFL) (SEQ ID NO:18) and sur11-19 (QPFLKDHRI) (SEQ ID NO:19). We were able to detect a strong spontaneous CTL response against the survivin derived peptide sur6-14 in PBL in a CLL patient and in a breast cancer patient (
(145) Summary of HLA Allele-Restricted Immune Responses to Survivin-Derived Peptides in Cancer Patients
(146) A range of survivin-derived peptides comprising 9-11 amino acid residues were tested for binding to the following HLA alleles: HLA-A1, HLA-A3, HLA-A11 and HLA-B7 using the assembly assay for peptide binding to MHC class I molecules described in the preceding examples. In addition, several of the peptides were tested for their capacity to elicit a CTL immune response using the ELISPOT assay as also described above.
(147) A summary of the results, including results obtained in the previous examples, are given in the below Table 4:
(148) TABLE-US-00006 TABLE4 C.sub.50andELISPOTdataforselectedsurvivin-derivedpeptides HLA Peptide SEQID allele length Position Sequence C.sub.50(M) Remarks NO: Footnotes HLA-A1 9mer Sur14-22 LKDHRISTF NB 22 Sur51-59 EPDLAQCFF NB 7 Sur38-46 MAEAGFIHC NB 23 Sur93-101 FEELTLGEF >100 24 10mer Sur34-43 TPERMAEAGF NB 20 Sur47-56 PTENEPDLAQ NB 25 Sur49-58 ENEPDLAQCF NB 26 Sur92-101 QFEELTLGEF 2 27 Control C1 VSDGGPNLY 0.8 28 peptide Modified sur14Y9 LKDHRISTY NB 29 peptides sur51Y9 EPDLAQCFY Weak 9 binding sur93Y9 FEELTLGEY NB 30 sur92Y9 QFEELTLGEY NB 31 sur34Y9 TPERMAEAGY NB 32 sur49Y9 ENEPDLAQCY NB 33 Sur92T2 QTEELTLGEF 2 34 Sur92S2 QSEELTLGEF 100 35 Sur93T2 FTELTLGEF 1 36 Sur93S2 FSELTLGEF 30 37 Sur38Y9 MAEAGFIHY 0.8 38 Sur47Y10 PTENEPDLAY 0.4 39 HLA-A2 Sur4-14 PTLPPAWQPFL NB 66 Sur18-28 RISTFKNWPFL 69 67 Sur54-64 LAQCFFCFKEL NB 68 Sur86-96 FLSVKKQFEEL 72 69 Sur88-98 SVKKQFEELTL NB 70 Sur103-113 KLDRERAKNKI NB 71 Control EBV,BMLF1 GLCTLVAML 3 72 peptide HIV,Pol ILKEPVHGV 0.2 73 HLA-A3 9mer Sur5-13 TLPPAWQPF NB 40 Sur53-61 DLAQCFFCF NB 41 Sur54-62 LAQCFFCFK NB 42 Sur95-103 ELTLGEFLK >100 43 Sur112-120 KIAKETNNK 2 44 i 10mer Sur13-22 FLKDHRISTF NB 45 Sur18-27 RISTFKNWPF NB 46 Sur53-62 DLAQCFFCFK 100 47 ii Sur84-93 CAFLSVKKQF NB 48 Sur101-110 FLKLDRERAK NB 49 Sur103-112 KLDRERAKNK NB 50 Sur112-121 KIAKETNNKK 1 51 Sur113-122 IAKETNNKKK NB 52 Control C3 ILRGSVAHK 0.1-0.3 53 peptide Modified Sur5K9 TLPPAWQPK 2 54 peptides Sur53K9 DLAQCFFCK NB 55 Sur54L2 LLQCFFCFK 1 56 Sur13K9 FLKDHRISTK NB 57 Sur18K10 RISTFKNWPK 0.02 58 Sur113L2 ILKETNNKKK >100 59 SurEx3-A3-1 TIRRKNLRK 0.5 60 iii SurEx3-A3-2 PTIRRKNLRK NB 61 Sur2b-A3-1 RITREEHKK NB 62 Control InfluenzaA, ILRGSVAHK 0.1 74 peptide nucleoprotein 265-273 HLA-A11 9mer Sur5-13 TLPPAWQPF NB 40 Sur53-61 DLAQCFFCF NB 41 Sur54-62 LAQCFFCFK 0.4 42 Sur95-103 ELTLGEFLK NB 43 Sur112-120 KIAKETNNK 1 44 10mer Sur13-22 FLKDHRISTF NB 45 Sur18-27 RISTFKNWPF NB 46 Sur53-62 DLAQCFFCFK 5 47 Sur84-93 CAFLSVKKQF NB 48 Sur101-110 FLKLDRERAK NB 49 Sur103-112 KLDRERAKNK NB 50 Sur112-121 KIAKETNNKK >100 51 iv Sur113-122 IAKETNNKKK NB 52 Control C4 AVFDRKSDAK 0.2 63 peptide HLA-B7 9mer Sur6-14 LPPAWQPFL >100 18 v Sur11-19 QPFLKDHRI >100 19 Sur46-54 CPTENEPDL NB 6 Sur51-59 EPDLAQCFF NB 7 10mer Sur34-43 TPERMAEAGF NB 20 Control C6 QPRAPIRPI 0.1 64 peptides C7 RPPIFIRRL 0.5 65 i An response was observed against the peptide Sur112-120 in one lymphoma patient (HEM34) by means of ELISPOT. ii Responses were detected against the peptide Sur53-62 in 3 lymphoma patients (HEM9, 11, 34) by means of ELISPOT. iv A weak response was observed in a melanoma patient (FM-TIL95) by means of ELISPOT. vii A response was observed against Sur112-121 in a melanoma patient (PM6), most evident in metastatic lymph-node suspension, and weaker in the TIL from primary tumor and PBL by means of ELISPOT. viii An response against the peptide Sur6-14 was observed in a CLL patient (CLL9), and a weaker response was observed in a lymphoma patient by means of elispot (HEM 21) (data not shown.
Example 5
Therapeutic Trial Procedures Using Survivin-Derived Peptides as Immunogens
(149) Summary
(150) Five heavily pretreated stage IV melanoma patients were vaccinated with the modified HLA-A2-restricted survivin epitope, namely the sur1M2 peptide, presented by autologous dendritic cells in a compassionate use setting. Four of the patients mounted strong T-cell response to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining revealed the infiltration of survivin reactive cells into both visceral and soft tissue metastases. Notably, vaccination associated toxicity was not observed. The data demonstrate that it is feasible to induce T-cell response against survivin, even in late stage melanoma patients, and that these vaccinations are well tolerated.
(151) Materials and Methods
(152) Patient Eligibility Criteria and Treatment Regimen
(153) All clinical procedures were in accordance with the Declaration of Helsinki and all patients provided informed consent prior to therapy. Stage IV cutaneous or uveal melanoma patients were eligible when their disease was progressive despite at least two different chemo-, immuno, or chemoimmunotherapies. In addition, a patients had to be 18 years or older, express HLAA*0201, and suffer from measurable disease validated by cranial, thoracic and abdominal computed tomography scans. Patients' Karnofsky index had to be 60% or better. No systemic chemo-, and/or immunotherapy was allowed within 4 week prior to vaccination. Important exclusion criteria were evidence of CNS metastases, active autoimmune or infectious diseases, pregnancy and lactation, as well as significant psychiatric abnormality. Peptide pulsed dendritic cells were generated as previously described (82). Briefly, PBMCs from leukapheresis were isolated on LYMPHOPREP (density gradient medium) (Nycomed Pharma), frozen in aliquots and stored in liquid nitrogen. One week prior to vaccination, PBMCs were thawed, washed and cultured in medium containing gentamycin, glutamine and heat inactivated autologous plasma. On day 1 and 5, IL-4 and GM-CSF were added. To differentiate mature DCs, TNF- and prostaglandin E2 were added on day 6. On day 7, cells displaying phenotypical and morphological characteristics of mature DCs, i.e. a veiled appearance and =75% CD83 expression, were pulsed with a modified survivin-derived HLA-A2 restricted survivin.sub.96-104 epitope, LMLGEFLKL (SEQ ID NO 10)(Clinalfa, Switzerland) 14. Cells were only used for vaccination if microbial tests of samples taken from cultures on days 1 and 5 proved to be sterile.
(154) Patients were vaccinated at 7-day intervals for the first two vaccinations followed by 28-day intervals for further vaccinations. A total of 10-2010.sup.6 mature, survivin.sub.96-104 pulsed DCs were resuspended in PBS, containing 1% human serum albumin, and injected intradermally in aliquots of 1.510.sup.6 DCs per injection site in the ventromedial regions of the thighs close to the regional lymph nodes. Limbs where draining lymph nodes had been removed and/or irradiated were excluded. Leukapheresis was repeated after 5 vaccinations in absence of severe deterioration of patient's state of health or occurrence of CNS metastases.
(155) Measurement of Clinical and Immunological Responses
(156) CT scans were performed prior to vaccination and every three months thereafter or in case of severe clinical signs of disease progression. Immunological responses were monitored by the ELISPOT assay, using PBMCs obtained every three months, to detect survivin.sub.96-104 specific IFN- release. To extend the sensitivity of the ELISPOT assay, PBMCs were stimulated once in vitro at a concentration of 110.sup.6 cells per ml in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Md.), supplemented with 5% heat-inactivated human serum and 2 mM of L-glutamine in the presence of 10 M of peptide. Two days later, 40 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) were added. After 10 days the cells were tested for reactivity. To this end, nitrocellulose bottomed 96-well plates (MULTISCREEN MAIP N45 (96-well assay plate), Millipore, Glostrup, Denmark) were coated with an anti-IFN-7 antibody (1-D1K, Mabtech, Sweden). Lymphocytes were added at 10.sup.4-10.sup.5 cells in 200 l X-VIVO medium (chemically defined, serum-free media) per well together with 10.sup.4 T2-cells and the relevant peptides at a final concentration of 2 M. After an overnight incubation at 37 C. and two washes, the biotinylated detection antibody (7-B6-1-Biotin, Mabtech, Sweden) was added; its specific binding was visualised using alkaline phosphatase-avidin together with the respective substrate (GibcoBRL). The reaction was terminated upon the appearance of dark purple spots, which were quantitated using the ALPHA IMAGER System (imaging software) (Alpha Innotech, San Leandro, Calif., USA).
(157) Survivin96-104/HLA-A*0201 reactive CD8+ T lymphocytes were also tracked in situ both at the vaccination sites as well as in visceral, soft tissue, or cutaneous metastases by means of multimeric survivin96-104/HLA-A*0201 complexes. Vaccination sites were excised 24 h after intradermal injection in all patients, whereas metastatic lesions were only removed in selected patients, if easily accessible (patients KN and GB), or removed during a curative intent (patient WW). The staining procedure for multimeric peptide/MHC complexes has been described recently (68). The multimeric survivin96-104/HLA-A*0201 complexes were generated by introduction of a recognition site for enzymatic biotinylation at the 5 end of the extracellular domains of HLA-A*0201 (residues 1-275). The recombinant protein was purified by size-exclusion (SEPHADEX G 25 (gel filtration), Pharmacia, Erlangen, Germany) and ion exchange (MONO-Q, Pharmacia) chromatography and folded in vitro by dilution in presence of the respective peptides and 2-microglobulin. After gel filtration on a Sephadex G25 (gel filtration) column, the protein was multimerized with streptavidin-FITC conjugated to dextran molecules (kindly provided by L. Winther, DAKO, Copenhagen, Denmark) to generate multivalent HLA-dextran complexes. Cryopreserved sections of the respective samples were dried over-night and subsequently fixed in cold acetone for 5 min. All incubation steps were performed in the dark at room temperature as follows: (i) 45 min of an anti-CD8 antibody (1:100, clone HIT8a, Pharmingen, San Diego, Calif.), (ii) Cy3-conjugated goat antimouse (1:500 diluted; code 115-165-100, Dianova, Hamburg, Germany) for 45 min and finally (iii) the multimers for 75 min. Between each step the slides were washed twice for 10 min in PBS/BSA 0.1%. Finally, slides were mounted in VECTASHIELD (antifade mounting medium) and observed under a Leica Confocal Microscope (TCS 4D, Leica, Mannheim, Germany).
(158) Results
(159) Patient Characteristics, Toxicity and Clinical Course
(160) Five far-advanced stage IV melanoma patients were enrolled, two suffering from uveal melanoma, one from soft tissue melanoma and the remaining two from cutaneous melanoma. Due to the manifestation of symptomatic brain metastases, one patient was taken off therapy after only two vaccinations. The other four patients received up to 15 vaccinations. One patient died from cardiac arrest in tumor free status after surgical resection of remaining metastases. Another patient was taken off therapy after 10 vaccinations because of appearance of visceral metastases (RW). One patient remained on study after 15 vaccinations. Detailed patient characteristics, previous therapy, number of vaccinations and survival status are summarized in table 5.
(161) No major toxicities occurred. Thus, hemoglobin, leucocytes and thrombocytes, as well as lactate dehydrogenase, creatinine and cholinesterase were not influenced by the vaccination therapy (
(162) Survivin-Specific CD8+ T Cell Responses
(163) To monitor the kinetics of cytotoxic T cell responses, PBMCs obtained prior to and three months after vaccination were tested for reactivity to the modified survivin.sub.96-104 epitope by ELISPOT for IFN-. Before analysis, PBMCs were stimulated once in vitro to extend the sensitivity of this assay. In all four patients tested, an induction of survivin.sub.96-104 reactive T cells was evident (
(164) The prognostic and clinical value of measurements of tumor-specific T-cell responses in peripheral blood has been questioned repeatedly; thus, we also tested for the presence of survivin.sub.96-104/HLA-A*0201 reactive CD8+ T lymphocytes among tumor infiltrating lymphocytes in situ by peptide/MHC multimer staining. To validate the method, we first analyzed tissue samples from delayed type hypersensitivity reactions occurring at the vaccination site within 24 hrs. This analysis confirmed earlier observations that intradermal injections of peptide-pulsed DC induce a strong peptide-specific inflammatory T-cell infiltrate. Subsequently, the peptide/MHC multimer staining procedure was applied on soft tissue and visceral metastases, which revealed the presence of survivin.sub.96-104/HLA-A*0201 reactive cells among the CD8+ infiltrate. This observation suggests that the vaccination does not only induce T cell with the desired specificity, but also endows them with the necessary homing capacity.
(165) TABLE-US-00007 TABLE 5 Summary of vaccination trials: patient characteristics Time from Survival Patient Age/ primary tumor Measurable No. of after first ID Sex to stage IV Previous therapy disease Clinical outcome vaccinations vaccination* GB 40/female 4 years, 8 LITT, fotemusine/IL- liver PD (slow growth of 15 +14 months months 2IFN, pre-existing and new treosulfan/gemcitabine hepatic lesions, new pancreas and pleural metastases) KN 53/male 11 years IL-2IFN/histamine, liver, kidney, PD (slow growth of 13 13 months fotemustine, soft tissue, bone pre-existing lesions, treosulfan/gemcitabine new lymph node, pleural and mediastinal lesions) WW 73/male 14 months surgery, DC- liver PD (stable hepatic, 12 12 months vaccination, but new adrenal due post- decarbazine metastasis) surgical stroke RW 72/male 16 years surgery, radiotherapy, soft tissue PD (growth of pre- 12 +12 months adriblastin/ifosfamid, existing and new soft ixoten, decarbazine, tissue metastases; TNF/melphalan detection of heart, lung and muscle metastases after 12 vaccinations) PB 52/male 2 years, 3 radiotherapy lung, kidney PD (new skin and 2 4 months months brain metastases)
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