Supplementary methods
Liquid Chromatography Mass spectrometry (LCMS). The HLA peptide pools as obtained were
separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPLC
system, Waters) and the eluting peptides were analyzed in an LTQ-Orbitrap hybrid mass
spectrometer (Thermo Fisher Scientific) equipped with an electrospray ionization (ESI) source. Eluted
peptide pools were loaded directly onto the analytical fused-silica micro-capillary column (75 µm i.d.
x 250 mm) packed with 1.7 µm C18 reversed-phase material (Waters) applying a flow rate of 400 nl
per minute. Subsequently, the peptides were separated using a two-step 180 minute-binary gradient
from 10% to 33% B at a flow rate of 300 nl per minute. The gradient was composed of Solvent A
(0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass
capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQOrbitrap mass spectrometer was operated in the data-dependent mode using a TOP5 and a TOP3
strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R =
30 000 for TOP3, R = 60000 for TOP5), which was followed by MS/MS scans either in the Orbitrap (R
= 7500) on the 5 most abundant precursor ions with dynamic exclusion of previously selected ions
(TOP5) or in the LTQ on the 3 most abundant precursor ions with dynamic exclusion of previously
selected ions (TOP3).
Data analysis and peptide sequence identification. The LCMS data was processed by analyzing the
LCMS survey (parent mass of the unfragmented peptide) as well as the Tandem-MS (MS/MS) data
(spectra of fragmented peptides containing sequence information). Data analysis was optimized and
adapted for identification of HLA peptides using the following methods. Each new data set is
integrated in the MySQL-based database. Tandem-MS spectra were extracted using msn_extract
(ThermoFischerScientific)
and
searched
with
Sequest
algorithm
(http://fields.scripps.edu/sequest/index.html) against the IPI database. The protein database hits
were subsequently validated by automated quality filtering using thresholds established on manually
1
identified HLA peptidomics data. For increased identification sensitivity, an in-house developed
spectral clustering algorithm was used to assign spectra to known peptide MS/MS clusters which are
being collected in the fragment spectra library. Before being considered as peptide vaccine
candidates, peptide sequences suggested by the described automated pipeline were confirmed by
manual inspection. The identity of peptides was further assured by comparison of the recorded
natural peptide fragmentation pattern of a synthetic reference peptide with the sequence in
question.
These
methods
are
also
detailed
in
two
patents
available
at
www.google.com/patents/US20050221350 and www.google.com/patents/US20110257890 .
Relative peptide quantification. LC-MS survey data (signals of intact and unfragmented peptides for
quantitative information) was analyzed independently of the Tandem-MS fragment spectra data
(recorded in the same experiment – and resulting in peptide sequence information) making use of
the high-mass accuracy. To extract LC-MS signals as well as the signal areas (ion counting) the
program SuperHirn (ETH Zürich) [Mueller et al. 2007] was used. Thus each identified peptide could
be associated with quantitative data allowing relative quantification between samples and tissues. To
account for variation between technical and biological replicates, a two-tier normalization scheme
was used based on central tendency normalization. The normalization assumes that most measured
signals result from house-keeping peptides and the small fraction of over-presented peptides does
not influence the central tendency of the data significantly. In the first normalization step replicates
of the same sample are normalized by calculating the mean presentation for each peptide in the
respective replicate set. This mean is used to compute normalization factors for each peptide and LCMS run. Averaging over all peptides results in run-wise normalization factors which are applied to all
peptides of the particular LCMS run. This approach ensures that systematic intra-sample variation is
removed, e.g. due to different injection volumes between replicate runs. Only peptides, which had a
coefficient of variation smaller than 50% between their replicate areas, were considered for
calculation of further normalization factors: Again the mean presentation of each peptide was
calculated, this time for all samples of a defined preparation antibody (e.g. BB7.2). The mean was
2
used to compute normalization factors for each peptide and sample. Averaging over all peptides
resulted in sample-wise normalization factors which were applied to all peptides of the particular
sample. Systematic bias due to different tissue weights or MHC expression levels was therefore
removed. For each peptide a presentation profile was calculated showing the mean sample
presentation as well as replicate variations. The profile juxtaposes GBM samples to a baseline of
normal tissue samples.
Identification and selection of HLA-A*02-restricted peptides. Assignment of HLA-A*02 restriction to
a peptide sequence was based on the following criteria: (i) detection by MS/MS analysis from a
sample immunoprecipitated with the HLA-A*02 specific antibody BB7.2 and (ii) SYFPEITHI score
analysis (for 9-mers and 10-mers) / anchor residue criteria in case that no SYFPEITHI matrix was
available. HLA-A*02-restriction was experimentally confirmed by determination of the binding
constant Kd for all ten selected peptides by an HLA refolding assay and by the fact that refolding of
HLA-A*02 monomers for tetramer staining as shown in Fig. 3 was successful for all ten peptides.
Analysis of GBM samples was performed until the rate of newly identified sequences among the last
1,000 total identifications dropped below 15% (ie. ≥85% of identifications resulted in already known
sequences of the GBM peptidome). For the 309 pre-selected peptides, median SYFPEITHI score is 25,
10% percentile is 17 – a score common to several published A*02 binding peptides
(www.syfpeithi.de). For 26 of the pre-selected peptides a SYFPEITHI score was not available (no 9- or
10mers), but they meet several characteristics of A*02 binding in terms of anchor residues.
Gene expression analysis. Gene expression analysis of all tumor and normal tissue RNA samples was
performed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide
microarrays (Affymetrix). The same normal kidney sample was hybridized to both array types to
achieve direct comparability of all samples. All steps were carried out according to the Affymetrix
manual
(http://media.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.p
3
df). Briefly, double-stranded cDNA was synthesized from 5–8 µg of total RNA, using SuperScript RTII
(Invitrogen) and the oligo-dT-T7 primer (MWG Biotech) as described in the manual. In vitro
transcription was performed with the BioArray High Yield RNA Transcript Labeling Kit (ENZO
Diagnostics, Inc.) for the U133A arrays or with the GeneChip IVT Labeling Kit (Affymetrix) for the
U133 Plus 2.0 arrays, followed by cRNA fragmentation, hybridization, and staining with streptavidinphycoerythrin and biotinylated anti-streptavidin antibody (Molecular Probes). Images were scanned
with the Agilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-Chip Scanner 3000 (U133
Plus 2.0), and data were analyzed with the GCOS software (Affymetrix), using default settings for all
parameters. Pairwise comparisons were calculated using the respective normal kidney array as
baseline. For normalization, 100 housekeeping genes provided by Affymetrix were used
(http://www.affymetrix.com/support/technical/mask_files.affx). Relative expression values were
calculated from the signal log ratios given by the software and the normal brain sample was
arbitrarily set to 1.0. An empirical mRNA over-expression score (S score) was calculated based on the
signal log ratios for each gene: S = 0.25x[meantumor-meannormal+meantumor-maxnormal+meantumor,top40%meannormal+meantumor,top40%-maxnormal]. S considers expression levels in the analyzed GBM samples
(average of all analyzed samples: meantumor) and average in 40% samples with highest expression
(meantumor,top40%) vs. average and highest expression in normal tissues. An empirical cut-off (S ≥ 1.8)
was set in order to pre-filter for “genes overexpressed”, qualifying the HLA-A*02-derived peptides
from these genes for more detailed analysis as potential targets for GBM immunotherapy.
Tissue Microarray, Immunohistochemistry and Immunofluorescent Stainings. TMA consisted of 250
formalin-fixed, paraffin-embedded GBM and 4 normal brain tissue samples as described elsewhere
[Campos et al. 2011]. Informed consent was obtained from each patient according to the research
proposals approved by the Institutional Review Board at Heidelberg Medical Faculty. Primary
antibodies used in our study were: anti-PTP-zeta (1:50), anti-NRCAM (1:200), anti-FABP7 (1:200),
anti-IGF2BP3 (uv, all Abcam), anti-NLGN4X (1:100), anti-Chi3L2 (1:25) anti-Brevican (1:200; SigmaAldrich), and anti-CSPG4 (1:500, Chemicon). Prior to TMA staining specificity of primary antibodies
4
was verified using corresponding isotype controls (all Acris) on glioma control tissues in equal
concentrations as primary antibodies and as indicated by the manufacturer. Antigen retrieval,
incubation with primary and secondary antibodies as well as detection with Vectastain Laboratories
ELITE ABC KIT (Vector Laboratories) was carried out as described [Campos et al. 2011]. Each tumor
biopsy was evaluated at 20x magnification by two independent investigators blinded to all clinical
data. Staining of TMA biopsies was semiquantitatively graded in an antigen-dependent manner
according to the estimated percentage of positive cells covering the whole tissue spot. In case of
inter-observer variability staining frequency on individual biopsies was counted manually. Average
staining patterns from all biopsies of an individual tumor were taken as final staining result.
Additional antibodies used for double immunofluorescence staining were: mouse monoclonal antihuman CD31 (1:100; both BD Pharmingen), anti-human GFAP (ready to use; PROGEN), anti-human
CD68 (1:25; Caltag) as well as secondary antibodies anti-mouse ALEXA488 and anti-rabbit ALEXA555
(1:500; both Invitrogen).
Peptides, recombinant MHC molecules, fluorescent tetramers and artificial APC. Peptides were
synthesized using standard Fmoc chemistry. The amino acid position and sequences of each peptide
are described in Table 1. The Melan-A peptide used as a control was Melan-A26-35 (EAAGIGILTV).
Biotinylated recombinant A*02 molecules and fluorescent MHC tetramers were produced as
described previously [Altman et al. 1996]. The costimulatory mouse IgG2a anti-human CD28 antibody
9.3 [Jung et al. 1987] was biotinylated using sulfo-N-hydroxysuccinimidobiotin as recommended by
the manufacturer (Perbio Science). For generation of artificial APCs, 5.6-µm-diameter streptavidincoated polystyrene particles with a binding capacity of 0.064 µg of biotin-FITC per mg of microsphere
(Bangs Laboratories) were resuspended at 5x106 particles per milliliter in PBS, 0.5% BSA with
biotinylated MHC (1 µg/ml) and anti-human CD28 antibody (3 µg/ml) and incubated at room
temperature for 30 min under agitation [Walter et al. 2003].
5
In vitro immunogenicity experiments. CD8+ T cells were stimulated with artificial APC. Briefly, CD8+ T
cell were isolated by magnetic bead positive selection using a MACS device (Miltenyi) and plated in
96-well round bottom plates in 100µl IMDM containing 8% human serum (Laboratoires Jacques Boy),
penicillin, streptomycin, non-essential amino acids, sodium pyruvate and Hepes (all from Invitrogen)
(CTL medium) at a concentration of 1x106 cells/well. Artificial APC (100µl) were added together with
rhIL-12 (5 ng/ml, Bioconcept). Cells were incubated for 3 days and medium was replaced with
addition of IL-2 (10 IU/ml) and IL-7 (2.5 ng/ml, Bioconcept). Cultures were restimulated similarly at
days 7 and 14 and tested at day 21 by flow cytometry. Each peptide was tested in 4 to 6 healthy
individuals (9-12 wells per peptide) and in 7 to 11 patients with GBM (2-5 wells per peptide). For
analysis of naïve and memory T cell populations, PBMC were sorted for CD8+ T cells by magnetic bead
selection. The CD8+ population was then stained with CD45RA and CCR7 antibodies (Beckman
Coulter) and the CD45RA+ CCR7+ (representing the naïve T cell population) and CD45RA- CCR7+/(representing the memory T cell population) fractions were sorted by FACS. Each population was
then separately stimulated with artificial APC incorporating either the BCA478-486 or the Melan-A26-35
control peptide three times as described above. Cultures were stained with MHC/peptide tetramers
incorporating either the cognate peptide or a control peptide.
6
Reference List
Altman JD, Moss PA, Goulder PJ et al. Phenotypic analysis of antigen-specific T lymphocytes. Science
1996; 274: 94-96.
Campos B, Bermejo JL, Han L et al. Expression of nuclear receptor corepressors and class I histone
deacetylases in astrocytic gliomas. Cancer Sci 2011; 102: 387-392.
Jung G, Ledbetter JA, Muller-Eberhard HJ. Induction of cytotoxicity in resting human T lymphocytes
bound to tumor cells by antibody heteroconjugates. Proc Natl Acad Sci U S A 1987; 84: 4611-4615.
Mueller LN, Rinner O, Schmidt A et al. SuperHirn - a novel tool for high resolution LC-MS-based
peptide/protein profiling. Proteomics 2007; 7: 3470-3480.
Walter S, Herrgen L, Schoor O et al. Cutting edge: predetermined avidity of human CD8 T cells
expanded on calibrated MHC/anti-CD28-coated microspheres. J Immunol 2003; 171: 4974-4978.
7
Table S1. List of the GBM-associated proteins identified by peptidomics
Gene
name
Accession
number
Description
Gene
name
Accession
number
Description
ACSBG1
Q96GR2
Long-chain-fatty-acid-CoA ligase ACSBG1
MAP1B
P46821
Microtubule-associated protein 1B
ACSL3
O95573
Long-chain-fatty-acid-CoA ligase 3
MAP2
P11137
Microtubule-associated protein 2
ADAM17
P78536
Disintegrin and metalloproteinase domaincontaining protein 17
MAP9
Q49MG5
Microtubule-associated protein 9
ADORA3
P33765
Adenosine receptor A3
MAP4K4
O95819
Mitogen-activated protein kinase kinase kinase
kinase 4
AP3B2
Q13367
AP-3 complex subunit beta-2
MBP
P02686
Myelin basic protein
APC
P25054
Adenomatous polyposis coli protein
MCM4
P33991
DNA replication licensing factor MCM4
ARAP2
Q8WZ64
Arf-GAP with Rho-GAP domain, ANK repeat
and PH domain-containing protein 2
MED27
Q6P2C8
Mediator of RNA polymerase II transcription
subunit 27
ARHGAP12
Q8IWW6
Rho GTPase-activating protein 12
MLC1
Q15049
Membrane protein MLC1
ARHGEF26
Q96DR7
Rho guanine nucleotide exchange factor 26
MSH2
P43246
DNA mismatch repair protein Msh2
ARMC8
Q8IUR7
Armadillo repeat-containing protein 8
MSH6
P52701
DNA mismatch repair protein Msh6
ARNT2
Q9HBZ2
Aryl hydrocarbon receptor nuclear
translocator 2
NAV2
Q8IVL1
Neuron navigator 2
ASNS
P08243
Asparagine synthetase
NCAN
O14594
Neurocan core protein
ATAT1
Q5SQI0
Alpha-tubulin N-acetyltransferase
NCAPG
Q9BPX3
Condensin complex subunit 3
ATP1A2
P50993
Sodium/potassium-transporting ATPase
subunit alpha-2
NCDN
Q9UBB6
Neurochondrin
ATP2B1
P20020
Plasma membrane calcium-transporting
ATPase 1
NDC80
O14777
Kinetochore protein NDC80 homolog
ATR
Q13535
Serine/threonine-protein kinase ATR
NES
P48681
Nestin
BCAN
Q96GW7
Brevican core protein
NLGN4X
Q8N0W4
Neuroligin-4, X-linked
BCAT1
P54687
Branched-chain-amino-acid aminotransferase
NMD3
Q96D46
60S ribosomal export protein NMD3
BCHE
P06276
Cholinesterase
NPAS3
Q8IXF0
Neuronal PAS domain-containing protein 3
C1QB
P02746
NR2E1
Q9Y466
Nuclear receptor subfamily 2 group E member 1
CACNA1A
O00555
NRCAM
Q92823
Neuronal cell adhesion molecule
CASK
O14936
Complement C1q subcomponent subunit B
Voltage-dependent P/Q-type calcium channel
subunit alpha-1A
Peripheral plasma membrane protein CASK
PCDH17
O14917
Protocadherin-17
CCDC88A
Q3V6T2
Girdin
PCDHGC3
Q9UN70
Protocadherin gamma-C3
CCDC93
Q567U6
Coiled-coil domain-containing protein 93
PDE4DIP
Q5VU43
Myomegalin
CCNB1
P14635
G2/mitotic-specific cyclin-B1
PDPN
Q86YL7
Podoplanin
CCND2
P30279
G1/S-specific cyclin-D2
PDS5A
Q29RF7
Sister chromatid cohesion protein PDS5 homolog A
CD163
Q86VB7
Scavenger receptor cysteine-rich type 1
protein M130
PGAP1
Q75T13
GPI inositol-deacylase
CENPF
P49454
Centromere protein F
PHKG1
Q16816
Phosphorylase b kinase gamma catalytic chain,
skeletal muscle isoform
CEP170
Q5SW79
Centrosomal protein of 170 kDa
PLEKHA4
Q9H4M7
Pleckstrin homology domain-containing family A
member 4
CHI3L1
P36222
Chitinase-3-like protein 1
PLIN2
Q99541
Perilipin-2
CHI3L2
Q15782
Chitinase-3-like protein 2
PLXNB3
Q9ULL4
Plexin-B3
CLIP2
Q9UDT6
PON2
Q15165
Serum paraoxonase/arylesterase 2
COG4
Q9H9E3
POSTN
Q15063
Periostin
CRMP1
Q14194
CAP-Gly domain-containing linker protein 2
Conserved oligomeric Golgi complex subunit
4
Dihydropyrimidinase-related protein 1
PRMT3
O60678
Protein arginine N-methyltransferase 3
CSPG4
Q6UVK1
Chondroitin sulfate proteoglycan 4
PRUNE2
Q8WUY3
Protein prune homolog 2
CSRP2BP
Q9H8E8
Cysteine-rich protein 2-binding protein
PTPRZ1
P23471
Receptor-type tyrosine-protein phosphatase zeta
CYBB
P04839
Cytochrome b-245 heavy chain
PUS7L
Q9H0K6
Pseudouridylate synthase 7 homolog-like protein
DCLK2
Q8N568
Serine/threonine-protein kinase DCLK2
PYGB
P11216
Glycogen phosphorylase, brain form
DOCK10
Q96BY6
Dedicator of cytokinesis protein 10
QKI
Q96PU8
Protein quaking
DPYSL3
Q14195
Dihydropyrimidinase-related protein 3
RB1
P06400
Retinoblastoma-associated protein
DPYSL4
O14531
Dihydropyrimidinase-related protein 4
SACS
Q9NZJ4
Sacsin
8
DTNA
Q9Y4J8
Dystrobrevin alpha
Proteasome-associated protein ECM29
homolog
Endothelin B receptor
SAMSN1
Q9NSI8
SAM domain-containing protein SAMSN-1
ECM29
Q5VYK3
SDC3
O75056
Syndecan-3
EDNRB
P24530
SEC31A
O94979
Protein transport protein Sec31A
EGFR
P00533
Epidermal growth factor receptor
Eukaryotic translation initiation factor 4
gamma 3
Elongation of very long chain fatty acids
protein 2
SEC61G
P60059
Protein transport protein Sec61 subunit gamma
EIF4G3
O43432
SERPINA3
P01011
Alpha-1-antichymotrypsin
ELOVL2
Q9NXB9
SEZ6L
Q9BYH1
Seizure 6-like protein
ENC1
O14682
Ectoderm-neural cortex protein 1
SFPQ
P23246
Splicing factor, proline- and glutamine-rich
EXOC1
Q9NV70
Exocyst complex component 1
SLC1A3
P43003
Excitatory amino acid transporter 1
FABP7
O15540
Fatty acid-binding protein, brain
SLC1A4
P43007
Neutral amino acid transporter A
FAM115A
Q9Y4C2
Protein FAM115A
SLC4A4
Q9Y6R1
FEN1
P39748
Flap endonuclease 1
SLCO1C1
Q9NYB5
GABPA
Q06546
GA-binding protein alpha chain
SMARCA1
P28370
GFAP
P14136
Glial fibrillary acidic protein
SMARCA5
O60264
Electrogenic sodium bicarbonate cotransporter 1
Solute carrier organic anion transporter family
member 1C1
Probable global transcription activator SNF2L1
SWI/SNF-related matrix-associated actindependent regulator of chromatin subfamily A
member 5
GFPT2
O94808
Glucosamine-fructose-6-phosphate
aminotransferase 2
SMC2
O95347
Structural maintenance of chromosomes protein 2
GLT25D2
Q8IYK4
Procollagen galactosyltransferase 2
SMC3
Q9UQE7
Structural maintenance of chromosomes protein 3
GPM6B
Q13491
Neuronal membrane glycoprotein M6-b
SMC6
Q96SB8
Structural maintenance of chromosomes protein 6
GPR56
Q9Y653
G-protein coupled receptor 56
SOCS6
O14544
Suppressor of cytokine signaling 6
GRIA2
P42262
Glutamate receptor 2
SPAG9
O60271
C-Jun-amino-terminal kinase-interacting protein 4
GRIA3
P42263
Glutamate receptor 3
SRP72
O76094
Signal recognition particle 72 kDa protein
H2AFY
O75367
Core histone macro-H2A.1
SRRT
Q9BXP5
Serrate RNA effector molecule homolog
HEATR6
Q6AI08
HEAT repeat-containing protein 6
SSX2IP
Q9Y2D8
Afadin- and alpha-actinin-binding protein
HP1BP3
Q5SSJ5
Heterochromatin protein 1-binding protein 3
STK17A
Q9UEE5
Serine/threonine-protein kinase 17A
ID3
Q02535
TLR7
Q9NYK1
Toll-like receptor 7
IGF2BP3
O00425
TMEM144
Q7Z5S9
Transmembrane protein 144
ITGB8
P26012
DNA-binding protein inhibitor ID-3
Insulin-like growth factor 2 mRNA-binding
protein 3
Integrin beta-8
TNC
P24821
Tenascin
KCTD3
Q9Y597
BTB/POZ domain-containing protein KCTD3
TOP2A
P11388
DNA topoisomerase 2-alpha
KIDINS220
Q9ULH0
Kinase D-interacting substrate of 220 kDa
TRIM23
P36406
E3 ubiquitin-protein ligase TRIM23
KIF1A
Q12756
Kinesin-like protein KIF1A
TRIM24
O15164
Transcription intermediary factor 1-alpha
KLHL7
Q8IXQ5
Kelch-like protein 7
TRIO
O75962
Triple functional domain protein
LANCL2
Q9NS86
LanC-like protein 2
TUBGCP5
Q96RT8
Gamma-tubulin complex component 5
LASS1
P27544
UBA6
A0AVT1
Ubiquitin-like modifier-activating enzyme 6
LPPR4
Q7Z2D5
VCAN
P13611
Versican core protein
MAGI2
Q86UL8
LAG1 longevity assurance homolog 1
Lipid phosphate phosphatase-related protein
type 4
Membrane-associated guanylate kinase, WW
and PDZ domain-containing protein 2
ZIC1
Q15915
Zinc finger protein ZIC 1
9
Table S2. Characteristics of the newly isolated GBM-associated antigens
Antigen selection criteria
BCA478-486
CHI10-18
CSP21-29
FABP7118-126
IGF2BP3552-560
NLGN4X131-139
NRCAM692-700
PTP195-203
PTP1347-1355
TNC3-11
Natural antigen presentation on GBM samples
Natural presentation
directly shown on tumor
samples
Antigen binding to HLA
Demonstrated high-affinity
binding to HLA-A2
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
90
40
100
40
90
95
100
100
45
X
X
X
X
X
X
+++
+++
+++
++
+++
++
mRNA overexpression of source protein
Over-expressed in GBM
80
samples (% samples)a
Over-expression in GBM
reported in literature
Antigen immunogenicity
In vitro immunogenicity
demonstratedb
X
++
+++
T cell responses against
source protein described
X
Relevant cancer-associated functions of source proteins
Oncofetal expression
X
pattern
Expression by brain cancer
stem cells
X
X
X
Pro-angiogenic effects/
Neovascularization
X
X
X
EGFR
Wnt
X
X
X
X
Biological properties of source protein
Sub-cellular locationd
ECM
EC
X
Wnt,
FGF2
X
X
Over-expression correlated
with higher tumor grade
Tumor-associated posttranslational modificationse
X
X
Link of cancer-associated
signaling pathwaysc
Over-expression linked to
decreased survival in GBM
+++
X
Roles in cell cycle
progression and cell
proliferation
Involvement in tumor
invasion, migration and
metastasis
++
X
X
X
X
X
CM
TM
TM
X
X
X
TM
CY
CY (NU)
TM
X
ECM
DG
a: number of samples with expression > expression on highest normal tissue, b: + in vitro immunogenicity detected; ++ >20% of tested wells and/or >=50% of tested
donors positive; +++ >50% of tested wells positive and/or >=80% of tested donors positive, c: EGFR = epithelial growth factor receptor; FGF2 = fibroblast growth factor
2; Wnt = Wnt / beta-catenin pathway (embryogenesis), d: CY = cytoplasmic; EC = extracellular localization; ECM = extracellular matrix; NU nuclear localization; TM =
transmembrane protein, e: DG deglycosylation
10