Process for the production of a DNA vaccine for cancer immunotherapy

Abstract

The present invention relates to a method for producing a DNA vaccine for cancer immunotherapy comprising at least the steps of (a) transforming an attenuated strain of Salmonella with at least one DNA molecule comprising at least one expression cassette encoding at least one antigen or at least one fragment thereof; (b) characterizing at least one transformed cell clone obtained in step (a); and (c) selecting at least one of the transformed cell clone(s) characterized in step (b) and further characterizing said at least one selected transformed cell clone. The present invention further relates to a DNA vaccine obtainable by the method according to the present invention.

Claims

1. A method of treating a human patient against cancer comprising administering to the patient a DNA vaccine comprising an attenuated strain of Salmonella with at least one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen or at least one fragment thereof, wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a, and wherein the at least one antigen comprises a neoantigen, wherein the neoantigen is a tumor-specific antigen that arises as a consequence of a tumor-specific mutation.

2. A method of treating a human patient against cancer comprising administering to the patient a DNA vaccine produced by a method comprising at least the following steps: a) transforming an attenuated strain of Salmonella with at least one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen comprising a neoantigen, wherein the neoantigen is a tumor-specific antigen that arises as a consequence of a tumor-specific mutation; b) characterizing at least one transformed cell clone obtained in step (a); c) selecting at least one of the transformed cell clone(s) characterized in step (b) and further characterizing said at least one selected transformed cell clone; wherein step (b) comprises at least one of the following substeps: bi) assessing the cell growth of at least one transformed cell clone obtained in step (a) over time; bii) assessing the stability of the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen in the at least one transformed cell clone obtained in step (a); biii) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen from at least one transformed cell clone obtained in step (a) and characterizing the at least one isolated DNA molecule by restriction analysis and/or sequencing; biv) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one from at least one transformed cell clone obtained in step (a), transfecting the at least one isolated DNA molecule into at least one eukaryotic cell and assessing the expression of the at least one antigen in said at least one eukaryotic cell; wherein step (c) comprises at least one of the following substeps: ci) assessing the number of viable cells per ml cell suspension of the at least one transformed cell clone selected in step (c); cii) assessing the stability of the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen in the at least one transformed cell clone selected in step (c); ciii) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen from the at least one transformed cell clone selected in step (c) and characterizing the at least one isolated DNA molecule by restriction analysis and/or sequencing; civ) isolating the at least one DNA molecule comprising at least one expression cassette encoding at least one antigen from the at least one transformed cell clone selected in step (c), transfecting the at least one isolated DNA molecule into at least one eukaryotic cell and assessing the expression of the at least one antigen in said at least one eukaryotic cell; cv) testing for the presence of bacterial, fungal and/or viral contaminants in at the least one transformed cell clone selected in step (c); cvi) verifying the bacterial strain identity of the at least one transformed cell clone selected in step (c); and wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a.

3. The method of claim 1, wherein the method is a personalized cancer immunotherapy.

4. The method of claim 3, wherein the neoantigen was shown to be expressed by tumor cells of the human patient.

5. The method of claim 4, wherein the neoantigen was identified by assessing the expression profile of the human patient's tumor either on mRNA or on protein level or by assessing pre-existing T cell immune responses to tumor antigens of the human patient.

6. The method of claim 1, wherein the DNA vaccine is administered orally.

7. The method of claim 1, wherein administering to the patient comprises administering to the patient (a) a first line treatment comprising a DNA vaccine comprising an attenuated strain of Salmonella with at least one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen or at least one fragment thereof, wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a, and wherein the at least one antigen or at least one fragment thereof is a tumor antigen and/or a tumor stroma antigen; and (b) a second line treatment comprising the DNA vaccine comprising an attenuated strain of Salmonella with at least one DNA molecule comprising at least one eukaryotic expression cassette encoding at least one antigen or at least one fragment thereof comprising a neoantigen, wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a.

Description

SHORT DESCRIPTION OF FIGURES

(1) FIG. 1: Overview over Drug Product manufacture

(2) FIG. 2: Flow chart of Drug Product manufacture

(3) FIG. 3: Expression plasmid synthesis

(4) FIG. 4: VXM04 Clones Growth Kinetics—OD.sub.600

(5) FIG. 5: VXM04 Clones Growth Kinetics—CFU/ml

(6) FIG. 6: VXM04 Clones Growth Kinetics—pH in Culture Medium

(7) FIG. 7: VXM08 Clones Growth Kinetics—OD.sub.600

(8) FIG. 8: VXM08 Clones Growth Kinetics—CFU/ml

(9) FIG. 9: VXM08 Clones Growth Kinetics—pH in Culture Medium

(10) FIG. 10: VXM01 Clones Growth Kinetics—OD.sub.600

(11) FIG. 11: VXM01 Clones Growth Kinetics—CFU/ml

(12) FIG. 12: VXM01 Clones Growth Kinetics—pH in Culture Medium

EXAMPLES

Example 1: Synthesis of Antigen/Antigen Fragment Encoding cDNA

(13) Synthesis of the cDNA inserts was performed by double strand in vitro gene synthesis. cDNAs encoding five different tumor antigens and one tumor stroma antigen were synthesized. The synthesized cDNAs are listed in Table 4.

(14) TABLE-US-00004 TABLE 4 Synthesized antigen cDNAs cDNA cDNA length Antigen type cDNA SEQ ID Human wild type 4071 bp Full length wild type SEQ ID NO 9 VEGFR-2 tumor stroma antigen Human wild type 1893 bp Full length wild type SEQ ID NO 11 MSLN tumor antigen Human wild type 2109 bp Full length wild type SEQ ID NO 12 CEA tumor antigen Wild type human 1683 bp Full length wild type SEQ ID NO 13 CMV pp65 tumor antigen K436N mutated 1683 bp Full length mutated SEQ ID NO 14 human CMV pp65 tumor antigen Truncated K436N 1608 bp Truncated mutated SEQ ID NO 15 mutated human tumor antigen CMV pp65

(15) The sequences of the cDNAs to be synthesized were subdivided into individual oligonucleotides of 40-50 bases. The designed oligonucleotides overlap and correspond to both DNA strands. These oligonucleotides were prepared by chemical synthesis. The in vitro synthesized forward and reverse oligonucleotides were combined in Eppendorf tubes and 5′-phosphorylated by incubation with T4 polynucleotide kinase and ATP. The phosphorylated forward and reverse oligonucleotides were denatured at 95° C. Complementary oligonucleotides were annealed by progressive cooling (1°/min) of the mixture. After the annealing process the aligned oligonucleotides were ligated using thermostable Taq DNA ligase. The denaturing and annealing process was repeated several times in a thermocycler to resolve mismatched base pairs and achieve complete matching of the complementary strands over the full length of the fragments. To increase the yield of the ligated fragments, PCR was performed after completion of the ligation step using primers annealing at outward positions of the fragments. The PCR amplification products were isolated by preparative agarose gel electrophoresis.

Example 2: Cloning of Antigen cDNA into Expression Plasmid

(16) The cDNAs synthesized in Example 1 were cloned into the plasmid pVAX10 via Nhel/Xhol.

(17) The thus generated recombinant plasmids were transformed into E. coli, isolated and sequenced. The complete sequences of the synthesized plasmids were determined and aligned to the corresponding reference sequences. The results of the sequence verification are summarized in Table 5.

(18) TABLE-US-00005 TABLE 5 Sequence verification of recombinant plasmids Identified mutations vs. cDNA reference sequence Human wild type VEGFR-2 none Human wild type MSLN 1 silent mutation Human wild type CEA none Wild type human CMV pp65 none K436N mutated human CMV pp65 none Truncated K436N mutated human CMV pp65 none

(19) One mismatch mutation was detected in the open reading frame for hMSLN at plasmid position 1392 (adenine instead of guanine) corresponding to position 657 in the cDNA. This mute mutation (wobble position) does not result in an altered consensus amino acid for MSLN. The mutated sequence was therefore accepted for transformation of S. typhi Ty21a and generation of the batch production clones. For all other plasmids the cloned sequences displayed 100% sequence identity to the reference sequences.

Example 3: Transformation of S. typhi Ty21a with Antigen Encoding Plasmids

(20) Salmonella typhi Ty21a was transformed with the five recombinant plasmids obtained in Example 2. For that purpose, single S. typhi Ty21a colonies were picked from agar plates and grown in 100 mL TSB culture medium overnight at 37° C. The cultures were then formulated with 15% sterile glycerol, aliquoted (1 ml), labelled, frozen, and stored at −75° C.±5° C. as Master Cell Bank, pending use. Two of the isolates prepared, designated VAX.Ty21-1 and VAX.Ty21-2, were selected for further use.

(21) The bacterial strain identity of the prepared isolates was verified by growing the isolates on bromothymol blue galactose agar and/or on Kligler iron agar. The characteristics of the obtained cell colonies used as Master Cell Bank is described in Table 6.

(22) TABLE-US-00006 TABLE 6 Characterization Testing of the Salmonella Typhi Ty 21a Isolates for Use as Master Cell Bank Test Result Parameter Test Method VAX.Ty21-1 VAX.Ty21-2 Identity BTB-Gal Agar Conforms, green to Conforms, green to yellowish colonies yellowish colonies without without discoloration of the discoloration of the medium medium Kligler Iron Agar Conforms, yellow Conforms, yellow coloration of the coloration of the medium, only little medium, only little gas formation gas formation Content CFU 7.6 × 10.sup.8 CFU/mL 7.0 × 10.sup.8 CFU/mL determination

(23) The isolate VAX.Ty21-1 was used as recipient strain for transformation with the recombinant plasmids generated in Example 2. The frozen glycerol stock of isolate VAX.Ty21-1 was streaked on LB agar plates (ACF soy peptone). One single colony was picked and cultivated in 3 ml of LB-medium (ACF soy peptone) overnight at 37° C. This culture was used to inoculate 2×300 ml LB-medium which was further incubated at 37° C. until the OD600 reached 0.5. In order to obtain competent cells for electroporation the culture was harvested by centrifugation at 4° C. The pellet was resuspended in 500 ml of ice cold H2O and centrifuged again. After two further washes in ice cold water/10% glycerol the pellet was resuspended in 2 ml of 10% glycerol (animal free), aliquoted (50 μl) and frozen on dry ice.

(24) For transformation one aliquot of competent cells per recombinant plasmid was thawed and electroporated with 3-5 μl of recombinant plasmid DNA each. Following a brief incubation period in 1 ml of LB (ACF) medium at 37° C., the cell suspensions were streaked on LB (ACF) agar plates containing kanamycin (25 and 50 μg/ml). The plates were incubated at 37° C. overnight.

(25) Three single colonies per transformation reaction were then selected and used to inoculate 3 ml of LB medium (ACF soy peptone) containing kanamycin (50 μg/ml). Cultures were incubated at 37° C. overnight. Plasmid DNA was isolated and plasmid identity was confirmed by restriction analysis.

(26) The selected clones were expanded in LB medium containing 50 μg/ml kanamycin. Cultures were mixed with 10% (v/v) glycerol, aliquoted (1 ml) and stored frozen at −70° C. The plasmids of the recombinant Ty21a clones were isolated and complete sequencing was performed. 100% sequence identity of the plasmids of each of the selected clones with the reference sequence was confirmed except for the hMSLN clone were one silent point mutation was identified (see Tab. 5).

(27) The generated transformed clones are listed in Table 7.

(28) TABLE-US-00007 TABLE 7 Transformed Clones cDNA Batch Production Clones Human wild type VEGFR-2 VXM01: VAX.11-01, VAX.11-02, VAX.21-01, VAX.21-02, VAX.21-03 Human wild type MSLN VXM04: VXM04_K06424, VXM04_K06425, VXM04_K06426 Human wild type CEA VXM08: VXM08h_K08.1.1, VXM08h_K08.2.2, VXM08h_K08.4.4 Wild type human CMV pp65 VXM65_1: h_VXM65_K_K65.3.3 K436N mutated human CMV VXM65_2: h_VXM65_N_K65.4.12 pp65 Truncated K436N mutated VXM65_3: h_VXM65_Nshort_K65.1.1 human CMV pp65

Example 4: Characterization of Transformed Cell Clones and Batch Production Clone Selection

(29) The following analytical parameters were evaluated for selection of the VXM01, VXM04 and VXM08 Batch Production Clones to be used for the establishment of the respective Drug Substances. Growth kinetics over time upon culturing in selective medium determined by OD.sub.600, pH and CFU Plasmid stability after cryo-conservation (% PS) Plasmid DNA extraction and confirmation of identity by plasmid restriction analysis Determination of antigen expression efficacy after transient transfection of plasmid DNA into a eukaryotic cell line

(30) The growth characteristics of the VXM01, VXM04 and VXM08 transformed clones listed in Table 7 were determined. All six VXM01 clones tested for growth expansion (VAX.11-01, VAX.11-02, VAX.21-01, VAX.21-02, VAX.21-03) grew well, but only clone VAX.11-02 grew to the same level as the Ty21a isolate from which it was derived. The growth characteristics of VXM04 clones VXM04_K06424, VXM04_K06425 and VXM04_K06426 are presented in FIG. 4, FIG. 5 and FIG. 6. All three clones displayed comparable growth rates in the culture medium with a slight growth advantage for clone VXM04_K06426. Regarding the VXM08 candidates (VXM08h_K08.1.1, VXM08h_K08.2.2 and VXM08h_K08.4.4), all clones displayed comparable growth characteristics, however, clone VXM08h_K08.1.1 was superior to the other two clones.

(31) Testing of the six VXM01 clones revealed that plasmid stability of VAX.11-02 was highest followed by VAX.11-03 and VAX.21-02. No significant difference was apparent between the three VXM04 clones with respect to plasmid stability before and after freezing. Testing of the three VXM08 clones revealed that plasmid stability of VXM08h_K08.4.4 was highest followed by VXM08h_K08.1.1 and VXM08h_K08.2.2.

(32) Restriction analysis of plasmid DNA isolated from each of the six VXM01 clones revealed the expected pattern of restriction fragments. Comparable amounts of plasmid DNA could be isolated from the three clones.

(33) Restriction analysis of plasmid DNA isolated from each of the three VXM04 clones revealed the expected pattern of restriction fragments. Comparable amounts of plasmid DNA could be isolated from the three clones.

(34) Restriction analysis of plasmid DNA isolated from each of the three VXM08 clones revealed the expected pattern of restriction fragments. Comparable amounts of plasmid DNA could be isolated from the three clones.

(35) After transfection of HEK293T cells with plasmid DNA isolated from the six VXM01 clones and Western blot analysis of cell extracts all six clones expressed the VEGFR-2 protein, with VAX.11-02, VAX.11-03 and VAX.21-02 showing the highest level, and with VAX.11-02 exhibiting a trend towards higher expression level according to visual inspection of the bands in the Western Blot gel.

(36) After transfection of HEK293T cells with plasmid DNA isolated from the three VXM04 clones and Western blot analysis of cell extracts three bands with apparent molecular weights of approximately 65 kDa, 40 kDa and 28 kDa were identified in each of the extracts. Based on staining intensity expression was highest when plasmid DNA isolated from clone 6316 was transfected.

(37) After transfection of HEK293T cells with plasmid DNA isolated from the three VXM08 clones and Western blot analysis of cell extracts all 3 clones expressed the glycosylated human CEACAM5 protein, with clone VXM08h_K08.1.2 showing the highest level, according to visual inspection of the bands in the Western Blot gel.

(38) Based on the data obtained from growth characteristics, plasmid stability, and protein expression studies, VAX.11-02 was selected as VXM01 Batch Production Clone for the preparation of the Drug Substance.

(39) After consideration of the data obtained from growth characteristics, plasmid stability, and protein expression studies, clone VXM04_K06426 was selected as VXM04 Batch Production Clone for the preparation of the Drug Substance.

(40) Based on the data obtained from growth characteristics, plasmid stability studies, clone VXM08h_K08.4.4 was selected as VXM08 Batch Production Clone for the preparation of the Drug Substance.

Example 5: Preparation and Release Testing of Drug Substances

(41) The VXM01, VXM04 and VXM08 Drug Substances were manufactured in compliance with GMP requirements starting with a single colony of the selected Batch Production Clone each. Several cell suspension dilutions per Batch Production Clone were plated on TSB agar plates containing 25 μg/ml kanamycin (Preculture 1). The plates were incubated at 37° C. for 20-30 h. Upon completion of the incubation time, three single colonies each were selected and transferred to three 50 ml flasks containing TSB medium plus 25 μg/ml kanamycin (Preculture 2). Colonies were grown to a maximum OD600 of <1.0 for 9 h t 1 h at 30° C. Agitation of each flask was set at 120 rpm. The flask with the highest OD value was selected for further cultivation. A volume of 50 ml of the Preculture 2 was transferred to a flask containing 1000 mL TSB medium plus 25 μg/mL kanamycin (main culture). After incubation at 30° C. for 9 h t 1 h, with agitation set at 180 rpm, the bacteria were grown to a target OD600 between 0.9 and 1.5. Once the fermentation was completed, glycerol was added to the culture to a final concentration of 15% (w/w). The suspension was mixed and then aliquoted (1 ml) into 2 ml cryovials. The vials were labelled and frozen immediately at −75° C.±5° C. for storage.

(42) The thus prepared Drug Substances VXM01, VXM04 and VXM08 were then further tested to establish the respective final Drug Products (release specification). The release characterization of the Drug Substances was based on the acceptance criteria listed in Table 3.

(43) 5.1 Biochemical Profile

(44) For direct plating of Ty21a and Drug Substances VXM01, VXM04 and VXM08 a loop of the completely thawed suspension was transferred to BTB-Gal-agar plates applying an appropriate streaking method to obtain single colonies. The inoculation of the control organism P. aeruginosa (ATCC 9027) and S. typhimurium (Moskau) was performed by transferring a bead of a Microbank® (storage system for microbial cultures) and subsequent streaking on the agar plate. For inoculation of KIA a loop (bead) of the same vial was first streaked onto the surface of the slant and then infeeded into the butt. The media were incubated for 48 h at 37° C.

(45) The resulting colonies showed the expected colony morphology. All three drug substances VXM01, VXM04 and VXM08 showed colony growth in the presence of 1.25% galactose on bromothymol blue galactose agar. The colonies were light-blue transparent and/or green to yellowish and did not result in colour change of the medium.

(46) 5.2 Serotyping

(47) A drop of antiserum (05 or 09) was transferred to a chamber slide. A loop of cell material of each Drug Substance to be tested was taken from the lower (wet) side of the KIA and placed next to the antiserum. The solutions were mixed with the loop. The resulting suspension was slightly turbid. The suspension was distributed by wiping of the chamber slides several times. The reactions were evaluated after 2 min against a black background.

(48) All three drug substances VXM01, VXM04 and VXM08 complied with the expected O9-positive and O5-negative serotype.

(49) 5.3 Restriction Analysis

(50) The recombinant plasmids were isolated from the Drug Substances VXM01, VXM04 and VXM08 and digested with two different digestion enzymes/combinations in separate reactions for an appropriate time. The reactions were stopped and analyzed on an agarose gel. The Endonucleases used for the restriction analysis are presented in Table 8.

(51) TABLE-US-00008 TABLE 8 Set-Up for Restriction Analysis Drug Restriction Substance Endonuclease Expected Size of Fragments (base pairs) VXM01 StyI 2209, 1453, 1196, 1094, 846, 623 and 159 VXM01 BamHI 7580bp VXM01 BgtI 2555, 2209, 1498 and 1318 VXM04 NheI/XhoI 1899 and 3494 VXM04 NdeI 1160 and 4233 VXM08 SacI 4142, 933 and 534 VXM08 BamHI 5609 VXM08 NheI/XhoI 3494 and 2115

(52) All three recombinant plasmids isolated from Drug Substances VXM01, VXM04 and WM08 showed the expected restriction pattern.

(53) 5.4 Sequence Analysis of the Plasmid

(54) The recombinant plasmids isolated from Drug Substances VXM01, VXM04 and VXM08 were quantified. After retransformation in E. coli, the recombinant plasmids were again isolated, quantified and sequenced.

(55) 100% sequence identity of the three recombinant plasmids with their respective reference sequence was confirmed.

(56) 5.5 Expression Analysis

(57) The recombinant plasmids were isolated from Drug Substances VXM01, VXM04 and VXM08 using a commercial DNA extraction and purification kit and the DNA content was determined. One day before transfection 7.5×10.sup.5 293 T cells were plated per well in 6-well plates to give a 90-95% confluence at the time of the assay. For transfection, the transfection complex consisting of the isolated plasmid DNA and Lipofectamine 2000™ was added to the cells and incubated for approximately 24 hours. After the incubation the cells were resuspended, washed once with PBS and lysed. Cell debris was pelleted by centrifugation. The supernatant was collected and protein content was determined. The samples were stored at ≤−70° C. until Western Blot analysis was, performed. The presence of the recombinant proteins was demonstrated and compared on a semi-quantitative basis with appropriate reference material.

(58) The expression levels of antigens VEGFR-2, MSLN and CEA were comparable to the chosen reference substance.

(59) 5.6 Viable Cell Number Determination

(60) Serial dilutions of bacterial suspensions of Drug Substances VXM01, VXM04 and VXM08 down to a dilution factor of 10.sup.−8 were prepared and plated onto agar plates. After appropriate incubation colonies were counted. Counting was started, when colonies were clearly visible, but not too large.

(61) The viable cell numbers determined are listed in Table 9.

(62) TABLE-US-00009 TABLE 9 Viable cell numbers Drug Substance Viable cell number VXM01   3 × 10.sup.8 CFU/ml VXM04 5.5 × 10.sup.9 CFU/ml VXM08 2.5 × 10.sup.9 CFU/ml
5.7 Plasmid Stability

(63) Serial dilutions of bacterial suspensions of Drug Substances VXM01, VXM04 and VXM08 (the same vials used for viable cell count testing) were prepared and plated onto TSB plates containing the antibiotic kanamycin. After appropriate incubation colonies were counted. Counting started, when colonies were clearly visible, but not too large. Plasmid stability was calculated by comparing colony forming units on TSB with and without kanamycin as follows:
PST=(CFU with kanamycin/CFU without kanamycin)×100

(64) All three Drug Substances VXM01, VXM04 and VXM08 complied with the pre-set plasmid stability acceptance criterion as specified in Table 3. The determined plasmid stability of all three recombinant plasmids was at least 75%.

(65) 5.8 Microbial Impurities

(66) To determine counts of total aerobic bacteria, molds and fungi and confirm the absence of the specific germs Escherichia coli, Salmonella sp., Pseudomonas aeruginosa, Staphylococcus aureus, and Clostridium sp. the Drug Substances VXM01, VXM04 and VXM08 were tested according to the European Pharmacopoeia Ph. Eur. monographs 2.6.12 and 2.16.13.

(67) All three Drug Substances VXM01, VXM04 and VXM08 complied with the pre-set microbial impurity acceptance criterion as specified in Table 3. In all three Drug Substances VXM01, VXM04 and VXM08 the total aerobic microbial count (TAMC) was not more than 10.sup.2 CFU/ml, the total yeast and mold count (TYMC) was not more than 2 CFU/ml and P. aeruginosa, S. aureus, E. coli, Clostridium sp. and other Salmonella strains were not detectable in 1 ml cell suspension.

(68) 5.9 Bacteriophage Testing

(69) The testing procedure for the detection of bacteriophages employed plating in soft-agar overlays containing an appropriate host and either the sample to be tested or a control suspension of phages. To improve the sensitivity of the assay a preceding enrichment step was included. In this step the samples were incubated for 4 h with appropriate host cells. Subsequently, one sample of each of these enrichment cultures was plated.

(70) All three Drug Substances VXM01, VXM04 and VXM08 complied with the pre-set purity of phage acceptance criterion as specified in Table 3. No phages were detectable in 100 μl of cell suspension after the enrichment step.

(71) TABLE-US-00010 SEQUENCE TABLE SEQ ID NO 1: expression plasmid SEQ ID NO 2: amino acid sequence VEGFR-2 SEQ ID NO 3: amino acid sequence WT1 SEQ ID NO 4: amino acid sequence MSLN SEQ ID NO 5: amino acid sequence CEA SEQ ID NO 6: amino acid sequence CMV pp65 SEQ ID NO 7: amino acid sequence CMV pp65 SEQ ID NO 8: amino acid sequence CMV pp65 SEQ ID NO 9: cDNA VEGFR-2 SEQ ID NO 10: cDNA WT1 SEQ ID NO 11: cDNA MSLN SEQ ID NO 12: cDNA CEA SEQ ID NO 13: cDNA CMVpp65 SEQ ID NO 14: cDNA CMVpp65 SEQ ID NO 15: cDNA CMVpp65