Production and characterization of amplified tumor-derived cRNA libraries to be used as vaccines against metastatic melanomas
© Carralot et al; licensee BioMed Central Ltd. 2005
Received: 22 June 2005
Accepted: 22 August 2005
Published: 22 August 2005
Anti-tumor vaccines targeting the entire tumor antigen repertoire represent an attractive immunotherapeutic approach. In the context of a phase I/II clinical trial, we vaccinated metastatic melanoma patients with autologous amplified tumor mRNA. In order to provide the large quantities of mRNA needed for each patient, the Stratagene Creator™ SMART™ cDNA library construction method was modified and applied to produce libraries derived from the tumors of 15 patients. The quality of those mRNA library vaccines was evaluated through sequencing and microarray analysis.
Random analysis of bacterial clones of the library showed a rate of 95% of recombinant plasmids among which a minimum of 51% of the clones contained a full-Open Reading Frame. In addition, despite a biased amplification toward small abundant transcripts compared to large rare fragments, we could document a relatively conserved gene expression profile between the total RNA of the tumor of origin and the corresponding in vitro transcribed complementary RNA (cRNA). Finally, listing the 30 most abundant transcripts of patient MEL02's library, a large number of tumor associated antigens (TAAs) either patient specific or shared by several melanomas were found.
Our results show that unlimited amounts of cRNA representing tumor's transcriptome could be obtained and that this cRNA was a reliable source of a large variety of tumor antigens.
The identification by van der Bruggen et al.  of the first tumor associated (TAA) antigen recognized by specific cytotoxic T lymphocytes (CTLs) in melanoma patients boosted the development of anti-cancer immunotherapy strategies. During the last years, vaccination protocols targeting differentiation antigens (MART-1/Melan-A [2, 3], gp100 , Tyrosinase [5, 6]) or cancer-testis antigens (MAGE [1, 7], NY-ESO1 ) were tested and showed encouraging results [9–11].
However, a growing body of evidence suggests that, instead of using defined antigens, targeting the whole spectrum of tumor antigens would represent an alternative, potentially more efficacious method [12–14]. Indeed, the use of total tumor material for vaccination allows the development of B and T cells directed against a large variety of known but also unknown TAAs . In addition, stimulating such a large spectrum of specific effectors directed against multiple epitopes restricted by diverse HLA class I and II types would reduce the risk of tumor escape through antigen loss or MHC downregulation [16–19]. Finally, another advantage of the whole tumor approach is that, in an autologous setting, patient's TAAs eventually stemming from tumor-specific somatic mutations could be targeted [20, 21].
In order to vaccinate patients with the whole spectrum of TAAs, several methods were developed. In 1998, Soiffer et al.  disclosed the results obtained by vaccinating patients with autologous irradiated tumor cells engineered to produce GM-CSF. The same year, Nestle et al.  showed partial or complete tumor remissions in six melanoma patients vaccinated with dendritic cells (DC) loaded with autologous tumor lysate. Alternatively, Boczkowski et al.  reported that mouse DCs pulsed in vitro with tumor RNA could trigger an anti-tumor immunity in vivo. Several groups further developed and optimized those different strategies [25–27] but faced the limitation imposed by the requirement of large amounts of tumor tissue for lysate preparation or for sufficient RNA yields extraction. In order to overcome this drawback, Boczkowski et al.  modified the SMART method (BD Biosciences Clontech, Palo Alto, CA) in order to in vitro transcribe tumor cDNA and performed therefore a one-step amplification of tumor mRNA. Transfected into antigen presenting cells (APCs), this amplified cRNA was shown in vitro to induce anti tumor immunity [29, 30]. As an alternative vaccination method, Hoerr et al.  demonstrated the capacity of mRNA coding for defined antigens or of total cRNA to trigger an antigen-specific immune response after direct intra-dermal injections of the ribonucleic acid. Similarly, Granstein et al.  showed protection against S1509 tumor cells in mice that received three intradermal injections of total RNA extracted from S1509 cells. Although still marginally studied compared to mRNA-loaded DC vaccines, the direct injection of mRNA represents a technology that offers the important advantage to circumvent the time and money consuming steps of generation of DCs.
Summary of mRNA libraries and clone analysis. In the case of MEL14, total RNA was extracted from ~5 × 104 pleural tumor cells (NA: Not applicable)
Weight of tumor sample (mg)
Quantity of extracted total RNA (μg)
Number of clones (cfu)
Size range of analyzed clones (nt)
Quantity of mRNA library prepared (mg)
Number of injection performed
1 × 105
500 – 4000
1 × 105
200 – 8000
5 × 105
250 – 1000
2 × 105
400 – 3500
2 × 105
500 – 1000
3 × 105
300 – 1200
5 × 105
500 – 1200
3 × 105
500 – 1200
4 × 104
350 – 800
2 × 105
600 – 1200
3 × 105
400 – 1000
6 × 104
400 – 1200
2 × 105
750 – 2000
1 × 105
400 – 10000
3 × 105
500 – 4000
2 × 105
450 – 3250
Whereas the SMART method was reported to maintain the relative levels of RNAs contained in the original transcriptome regardless of their size or their baseline expression , the cloning step in E. coli was on the contrary described to introduce a bias favoring short fragments . We thus analyzed the quality of the produced amplified-mRNA libraries to be used as a vaccine in melanoma patients. Several clones randomly picked-up within the produced libraries were analyzed by PCR and sequenced. In addition, the gene expression profiles of two metastases were compared to their corresponding cRNA-libraries.
Results and discussion
Tumor-derived mRNA library quality
Relative representation of transcripts
Patient-specific gene expression
Several tumor antigens are present in the cRNA libraries
List of the thirty transcripts showing the highest fluorescence signals in MEL02 amplified cRNA library
Ribosomal protein L23a
Eukaryotic translation elongation factor 1 alpha 1
Ribosomal protein S3A
RNase A family, 1 (pancreatic)
Peptidylprolyl isomerase A, cyclophilin A
Overexpressed in several cancers 
Ribosomal protein S23
Ribosomal protein L39
Melanoma differentiation antigen 
Ribosomal protein L31
Cytochrome c oxidase subunit VIc
Overexpressed in carcinomas 
Ribosomal protein L7
Overexpressed in gliomas 
Ribosomal protein L37a
Ribosomal protein S29
Secreted phosphoprotein 1, osteopontin
Important for tumorgenesis 
Overexpressed in several cancers 
Ribosomal protein S11
"Ribosomal protein S4, X-linked"
Nascent-polypeptide-associated complex alpha
Overexpressed in gliomas 
Ribosomal protein L23a
Involved in tumor proliferation 
ATP synthase, mitochondrial F0 complex, subunit g
Tubulin, alpha, ubiquitous
Overexpressed in breast cancers 
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex
Ribosomal protein S27a
Overexpressed in breast cancers 
H2A histone family, member Z
Overexpressed in several cancers 
SRY (sex determining region Y)-box 4
Overexpressed in lung cancers 
ATP synthase, mitochondrial F1 complex, epsilon subunit
Tumor protein, translationally-controlled 1
Involved in malignant transformation 
Cytochrome c oxidase subunit VIIa polypeptide 2
Related to sustained proliferation 
In order to vaccinate metastatic melanoma patients with autologous amplified tumor-derived cRNA, fifteen libraries were produced using a modified SMART method. Despite a heterogeneous amplification of tumor genes, this method provided us with an unlimited source of tumor and patient specific TAAs. Indeed, the microarray analysis of the libraries indicated the presence of high copy numbers of well-known tumor associated antigens such as Melan-A but also of abundant tumor-related antigens scarcely targeted in immunotherapy. Although not addressed in the present work, this method might also allow the targeting of tumor-specific mutations. These features makes of the amplification of tumor mRNA the method of choice to easily obtain unlimited amounts of RNA coding for patient's specific TAAs that can be applied as anti-tumor immunotherapy.
Materials and methods
Immediately after surgery, metastatic tissues from fully informed patients (Ethic committee approval Nr.: 269/2002) were chopped in ~0,1 cm3 pieces, and submerged in RNAlater solution from Ambion (Hungtingdon, UK), and stored at 4°C until histological identification as melanoma by an experienced pathologist.
Tumor total RNA extraction
Total RNA was extracted from tumors using the RNeasy mini kit from Qiagen (Hilden, Germany) following the instructions of the provider. Briefly, 15 to 30 mg samples placed in a 2 ml eppendorf tube were snap-frozen in a liquid nitrogen bath and disrupted with micropistils from Eppendorf (Hamburg, Germany). Tumor powder was resuspended in RLT buffer, homogenized through a 20-gauge needle and digested with 200 μg of proteinase K (Qiagen) at 55°C during 10 min. Samples were then clarified, loaded on RNeasy mini columns, washed and finally eluted in 50 μl of RNAse-free water. RNA was quantified by U.V spectrophotometry (O.D260/O.D280 ratio was over 1.8 in all cases) and analyzed on a 1,2% formaldehyde/agarose gel.
cDNA library generation
cDNA libraries of tumor total RNA were generated using the slightly modified Creator™ SMART™ PCR cDNA library construction kit from BD Biosciences Clontech (Heidelberg, Germany). Briefly, 1 μg of total RNA was reverse transcribed using SMART IV™ and CDS III/3' oligo-dT primers provided by the manufacturer. After termination of the reaction, 2 μl of cDNA were amplified using the Advantage 2 PCR kit (BD Biosciences Clontech). DNA polymerase was then inactivated with proteinase K and the cDNA library was digested with 200 U of Sfi I enzyme. cDNA libraries were then gel-purified on an 1% agarose gel and fragments from 300 bp to 10 kbp were extracted using E.Z.N.A.™ Gel Extraction kit from Peqlab GmbH (Erlangen, Germany). After precipitation, the cDNA library was ligated to dephosphorylated Sfi I-digested RNactive™ vector provided by CureVac GmbH (Tübingen, Germany) in three separated reactions to optimize vector/insert ratios.
The three ligation products were used to transform XL10-Gold ultracompetent cells from Stratagene (Heidelberg, Germany). For analysis, 1 and 10 μl of transformation broth were plated on 2 LB-ampicillin agar plates and, after overnight culture at 37°C, the number of clones was counted. The inserts of 8 clones per transformation were amplified by PCR using primers flanking the insertion sites and amplicons were analyzed on a 1% agarose gel. Libraries having more than 104 clones/ml and less than 20% of non recombinant clones, were amplified in three 300 ml maxicultures in 2X LB-ampicillin medium during 20 h at 33°C in order to limit uneven amplification of clones.
DNA preparation and linearization
Maxicultures were pooled, centrifuged down at 5 000 rpm for 10 min and plasmid DNA was extracted using EndoFree Plasmid Maxi (Qiagen). After precipitation, 100 μg of cDNA library were digested with 100 U of Not I enzyme. After phenol/chloroform extraction and ammoniumacetate precipitation, linearized cDNA libraries were resuspended in RNAse-free water, quantified by U.V. spectrophotometry (O.D260/O.D280 ratio was over 1.8 in all cases) and analyzed on 1% agarose gel.
cRNA In vitrotranscription
Twenty to hundred micrograms of linear cDNA library were in vitro transcribed using T7 mRNA Optikit from CureVac GmbH. After mRNA synthesis, DNA template was digested with 40 to 100 U of recombinant DNAse I purchased from Ambion. mRNA was then LiCl precipitated, phenol/chloroform purified, NaCl precipitated, and finally resuspended in PBS. cRNA was filter sterilized (0,2 μm), heat denatured at 80°C for 10 min before final sterile aliquoting. cRNA was quantified by U.V spectrophotometry (O.D260/O.D280 ratio was over 1.8 in all cases) and analyzed on 1.2% formaldehyde/agarose gel. Sterility of cRNA was checked by inoculating LB medium (in all cases, no bacterial growth was observed after 4 days at 37°C) and endotoxin content was determined using Bio-Whittaker (Verviers, Belgium) LAL assay kit (endotoxin content was always below 7 EU/ml).
For each library, 3 colonies per transformation were randomly picked-up with a pipette tip and used to inoculate 3 ml of LB-ampicillin medium. After overnight culture at 37°C, plasmid DNA was extracted using E.Z.N.A miniprep kit (Peqlab). Clone sequencing was performed using the ABI Big Dye and a T7 promoter primer. Sequences were purified on Autoseq. G-50 columns (Amersham Pharmacia Biotech, Freiburg, Germany), run on a 310 Genetic Analyzer from ABI PRISM™ (Applied Biosystems, Darmstadt, Germany) and analyzed with the Sequencing Analyzing 3.4.1 software (ABI PRISM). Finally, the BLAST algorithm  was used to identify matches to known genes.
Expression analysis of total tumor RNA and amplified tumor cRNA was performed on HG-U133A microarrays from Affymetrix (High Wycombe, UK) according to the manufacturer's eukaryotic sample and array processing standard procedure , which is based on the IVT method originally described by Van Gelder et al. . Briefly, 1st-strand cDNA synthesis was performed using an oligo(dT)24 primer containing a T7 promoter sequence. After RNA template degradation and cDNA's second strand cDNA synthesis, complementary RNA (cRNA) was transcribed in vitro using biotinylated NTPs and T7 RNA polymerase. After purification using RNeasy columns (Qiagen), 18 μg of biotin-labeled cRNAs were fragmented by metal-induced hydrolysis. Hybridization, staining, and scanning of microarrays were performed by the Microarray Facility Tübingen. Scanned images were processed using the Microarray Analysis Suite 5.0 (MAS 5.0; Affymetrix) and expression differences between tumor and library samples were determined by baseline comparison algorithms provided by the software. Data were further processed using Microsoft Access™ and Excel™.
JPC is supported by a "Fortüne" grant from the University of Tübingen and JP is supported by the DFG Graduiertenkollegue "Infektionsbiologie" of Tübingen.
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