ORIGINAL ARTICLE CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells Cheryl E. Myers • Paul Hanavan • Kwasi Antwi • Daruka Mahadevan • A. Jamal Nadeem • Laurence Cooke • Adrienne C. Scheck • Zachary Laughrey • Douglas F. Lake Received: 24 March 2010 / Accepted: 9 May 2011 / Published online: 29 May 2011 Ó Springer-Verlag 2011 Abstract Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self- proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGL TPKVTPS) corresponding to a frameshift in the 3 0 beta- hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA- A*0201. We confirmed this prediction using an HLA- A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti- peptide and CTL capable of killing GBM in vitro. Wild- type peptides as well as OPL induced anti-peptide CTL as measured by IFN-c ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further sub- stantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization. Keywords Cytotoxic T lymphocytes Optimized peptide ligand Glioblastoma multiforme Introduction Nearly 11,000 individuals were diagnosed with glioblas- toma multiforme (GBM) in 2009. Statistics suggest that less than 30% of those patients will be alive 5 years after diagnosis. Treatment for GBM includes neurosurgery, chemotherapy, and radiotherapy, but the tumor almost always recurs, likely due to chemo and radio-resistant stem cells and/or the inability to resect the entire tumor due to its location [1, 2]. Obviously, additional studies and approa- ches directed toward cure and therapy are needed for patients with GBM. Although many tumor antigens have been discovered and characterized [3], there remains a need to identify more tumor antigens due to the genetic instability and hetero- geneity of tumors. Over-expressed, tissue-specific, and mutated proteins each have potential to harbor peptide epitopes that could stimulate anti-tumor CTL. Character- ization of peptides that bind to major histocompatibility complex (MHC) class I has revealed that most peptides are presented to CD8 T cells as 8–10 amino acid peptides. However, identification of longer class I T-cell epitopes has been reported [4–6]. HLA-A*0201 (HLA-A2)-binding peptides are typically 8–10 amino acids in length with C. E. Myers P. Hanavan K. Antwi D. F. Lake (&) School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA e-mail: [email protected]Z. Laughrey Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA D. Mahadevan A. J. Nadeem L. Cooke Department of Medicine, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA A. C. Scheck Barrow Neurological Institute of SJHMC, Phoenix, AZ 85013, USA 123 Cancer Immunol Immunother (2011) 60:1319–1332 DOI 10.1007/s00262-011-1032-4
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CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells
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ORIGINAL ARTICLE
CTL recognition of a novel HLA-A*0201-binding peptide derivedfrom glioblastoma multiforme tumor cells
Cheryl E. Myers • Paul Hanavan • Kwasi Antwi • Daruka Mahadevan •
A. Jamal Nadeem • Laurence Cooke • Adrienne C. Scheck • Zachary Laughrey •
Douglas F. Lake
Received: 24 March 2010 / Accepted: 9 May 2011 / Published online: 29 May 2011
� Springer-Verlag 2011
Abstract Genetic instability of tumor cells can result in
translation of proteins that are out of frame, resulting in
expression of neopeptides. These neopeptides are not self-
proteins and therefore should be immunogenic. By eluting
peptides from human glioblastoma multiforme (GBM)
tumor cell surfaces and subjecting them to tandem mass
spectrometry, we identified a novel peptide (KLWGL
TPKVTPS) corresponding to a frameshift in the 30 beta-
hydroxysteroid dehydrogenase type 7 (HSD3B7) gene.
HLA-binding algorithms predicted that a 9-amino acid
sequence embedded in this peptide would bind to HLA-
A*0201. We confirmed this prediction using an HLA-
A*0201 refolding assay followed by live cell relative
affinity assays, but also showed that the 12-mer binds to
HLA-A*0201. Based on the 9-mer sequence, optimized
peptide ligands (OPL) were designed and tested for their
affinities to HLA-A*0201 and their abilities to elicit anti-
peptide and CTL capable of killing GBM in vitro. Wild-
type peptides as well as OPL induced anti-peptide CTL as
measured by IFN-c ELISPOTS. These CTL also killed
GBM tumor cells in chromium-51 release assays. This
study reports a new CTL target in GBM and further sub-
stantiates the concept that rational design and testing of
multiple peptides for the same T-cell epitope elicits a
broader response among different individuals than single
peptide immunization.
Keywords Cytotoxic T lymphocytes � Optimized peptide
ligand � Glioblastoma multiforme
Introduction
Nearly 11,000 individuals were diagnosed with glioblas-
toma multiforme (GBM) in 2009. Statistics suggest that
less than 30% of those patients will be alive 5 years after
diagnosis. Treatment for GBM includes neurosurgery,
chemotherapy, and radiotherapy, but the tumor almost
always recurs, likely due to chemo and radio-resistant stem
cells and/or the inability to resect the entire tumor due to its
location [1, 2]. Obviously, additional studies and approa-
ches directed toward cure and therapy are needed for
patients with GBM.
Although many tumor antigens have been discovered
and characterized [3], there remains a need to identify more
tumor antigens due to the genetic instability and hetero-
geneity of tumors. Over-expressed, tissue-specific, and
mutated proteins each have potential to harbor peptide
epitopes that could stimulate anti-tumor CTL. Character-
ization of peptides that bind to major histocompatibility
complex (MHC) class I has revealed that most peptides are
presented to CD8 T cells as 8–10 amino acid peptides.
However, identification of longer class I T-cell epitopes has
been reported [4–6]. HLA-A*0201 (HLA-A2)-binding
peptides are typically 8–10 amino acids in length with
C. E. Myers � P. Hanavan � K. Antwi � D. F. Lake (&)
School of Life Sciences, Arizona State University, Tempe, AZ
peptide (KLWGLTPKVTPS) showed an IC50 of 32 uM for
HLA-A2 on the T2 cell surface, 3-fold better than KLWG-
9 of 110 uM. OPL2 (FLFGLTPKV) demonstrated the best
relative affinity among the 4 peptides tested with an IC50 of
17 uM. Based upon the relative affinity results alone, one
might predict that OPL2 would elicit more active CTL
compared with wild-type KLWG-9. However, we have
observed in previous studies that optimized peptides with
the best affinity do not always elicit the most active CTL
from the most individuals [31]. Therefore, we first modeled
the peptides in HLA-A*0201 and then raised CTL on
KLWG-9, OPL1, and OPL2 peptides followed by testing
their reactivities against wild-type KLWG-9 and KLWG-
12 peptides in cytotoxicity assays.
Modeling of KLWG Peptides in HLA-A*0201
Since KLWG-12 bound to HLA-A*0201 with a better
relative affinity than KLWG-9, we modeled KLWG-9,
KLWG-12, OPL1, and OPL2 in the binding cleft of HLA-
A*0201 in an attempt to observe the potential reason(s) for
the apparent increased affinity of the 12-mer (Fig. 4). The
‘‘Total Score’’ value (Table 2) generated by the modeling
software (higher scores indicate stronger binding) for
KLWG-9 peptide mirrors the IC50 data. Modeling suggests
that KLWG-9 is the poorest binder among the four peptides
essentially due to lack of aromatic residues in P1 like
phenylalanine which are preferred [9, 14]. For KLWG-9,
the amine group in the K at P1 forms a salt bridge with E63
on HLA-A2 and an H-bond with the backbone –C=O of
E58 and Y59, while P3 (W) forms an H-bond with –C=O
backbone of Q155 (Fig. 4a). Residues 5–8 are generally
oriented toward solvent, similar to other HLA-A*0201-
binding peptides.
Although no crystal structures have been generated with
[10 amino acids bound to HLA molecules, our docking
models clearly demonstrate that a 12-mer can bind in the
same orientation as the KLWG-9-mer, and ranks second
best in IC50 measurements and ‘‘Total Score’’ modeling
values among other peptides tested (Table 2). The 12-mer
KLWGLTPKVTPS essentially maintains a similar mode of
binding as KLWG-9 with the N-terminal –NH2 group
forming an H-bind with –OH of Y7 and Y171 on the MHC
molecule. P1 (K) forms an H-bond with –C=O backbone of
Y59 and salt bridge with E63 on the MHC molecule, like
KLWG-9 (Fig. 4b). Unlike KLWG-9, P3 (W) points into
solvent and most likely interacts with the T-cell receptor,
while P8 (K) forms a salt bridge with Q72. Also unlike
KLWG-9, threonine at P6 is oriented away from the
binding groove, toward solvent, providing a potential Tcr
interaction. The two prolines (P7 and P11) provide a
scaffold for further peptide-protein backbone interactions,
but may also interact with Tcr. P10-12 appear stacked such
Fig. 1 MS/MS spectrum of KLWG-12. y and b ions from peptide
fragmentation are labeled along with the mass of each fragment
Table 1 continued
Epitope SYFPEITHIscore Protein name
AKLLTIPQTLLNIS [14] KLLTIPQTLL 23 Zinc finger protein 493
AKLLTIPQTLLNIS [14] LTIPQTLLNI 23 Zinc finger protein 493
PVDTRAKVVLPPSLPRA [17] VLPPSLPRA 22 Uncharacterized protein KIAA1522
ALRGAGGGGVRDPGRLH [17] ALRGAGGGGV 25 Latent-transforming growth factor beta-binding protein
4 (LTBP-4)
For both T98g and CRL2610, multiple peptide fragments were identified from the same protein, so the longest parent fragment is listed along with
predicted HLA-binding epitopes contained within the parent sequence. SYFPEITHI scores below 20 were not considered
Cancer Immunol Immunother (2011) 60:1319–1332 1325
123
that threonine (P10) and serine (P12) form salt bridges with
K146 on HLA-A*0201, which could be responsible for the
increased relative affinity observed in T2 cell assays.
The molecular models of the docked FLWGLTPKV
(OPL1) and FLFGLTPKV (OPL2) peptides are similar
(Fig. 4c, d). P1 (F) interacts through a pi-pi aromatic
interaction with W167. The N-terminal NH2 group
H-bonds to the –OH group of Y7 and Y171, while F at P3
forms an aromatic interaction with Y159. Like KLWG-9,
P2 (L) and P9 (V) are buried in hydrophobic pockets of
the MHC. P7 (P) acts as a linker between the N- and
C-terminal portions of the peptide, and could interact with
Tcr. There are 6 H-bonds between the peptide backbone
and MHC-I clustered in the C-terminal area of the pep-
tide. All other H-bonding interactions occur with the
peptide backbone clustered at the C-terminal end of the
peptide. These modeling studies suggest that by simply
having more peptide-MHC contact, affinity may increase
if the peptide still fits into the binding groove. However,
it is important not to over-optimize a peptide for MHC
binding such that few residues are available to interact
with Tcr [32].
CTL generation and IFN-c ELISPOT assay
Human PBMC were cultured with repeated weekly stim-
ulations of either KLWG-9 or OPLs to determine whether
Fig. 2 PCR amplification of
HSD3B7 exons 2-4. a Oligo-
dT-primed cDNA from brain
tumor cell lines lane 1, CRL-
2610; lane 2 T98g; lane 3,
U251; lane 4, U-87. b Oligo-dT-
primed cDNA from primary
GBM line in lane 1, Ao2V4;
GBM tumor tissue lane 2, LB;
lane 3, LH; lane 4, NN; lane 5,
OJ; lane 6, TL. Lane 7 (SN) is
oligo-dT-primed cDNA from
normal brain tissue. In (a) and
(b), b-actin cDNA (189 bp) was
amplified across two exons as a
control for genomic
contamination. If genomic DNA
was present, a 600-bp band
would be observed
corresponding to an intron
between exons
Fig. 3 Detection of refolded HLA-A*0201 onto peptide beads.
a HLA-A*0201-positive control peptide; b HLA-A*0201-negative
control peptide; c KLWG-12 peptide; d KLWG-9 peptide
Table 2 Relative (IC50) and predicted affinities of OPL for HLA-A2
Computer
modeling
Peptide Sequencea SYFPEITHIb IC50
lMcTotal scored
KLWG-12 KLWGLTPKVTPS n/a 32 7.0
KLWG-9 KLWGLTPKV 30 110 3.0
OPL1 FLWGLTPKV 30 76 4.8
OPL2 FLFGLTPKV 30 17 13.4
a Standard single letter amino acid code and residues shown in bold represent
deviations from KLWG-9b SFXC-based total score and binding affinity for the variant-RT peptide (I1Y)
and the twenty-six peptides bound to the HLA-A*0201 crystal structurec Peptide epitope prediction values calculated using SYFPEITHId SFXC based total score and binding affinity for the Variant-RT peptide (I1Y)
and the twenty-six peptides bound to the HLA-A*0201 crystal structure
1326 Cancer Immunol Immunother (2011) 60:1319–1332
123
the peptides elicited a CTL response (Table 3). Because we
eluted KLWG-12 from GBM tumor cells, we hypothesized
that CTL raised against the 9-mer and the OPLs as pre-
sented by a functional APC should cross-react with tumors
expressing KLWG-12. To address this hypothesis, CTL
were raised on KLWG-9 peptide and the OPLs and evalu-
ated for recognition of native KLWG-12 peptide as well as
the cognate KLWG-9 in IFN-c ELISPOT assays. Each of
five donors responded to the peptides, but with different
profiles (Table 3). For donor ND51, KLWG-9 peptide
elicited nearly equivalent numbers of anti-KLWG-12 and
KLWG-9 CTL: 439 and 443 spots per 5 9 104 PBMC,
respectively. ND 51 responded only moderately to KLWG-
12 and KLWG-9 after stimulation with OPLs. In contrast,
CTL from donor ND78 raised on OPL1 peptide responded
better than wild-type and OPL2 peptide to the KLWG-12
and KLWG-9 target peptides with 97 and 105 spots per
5 9 104 PBMC, respectively. We were unable to raise
significant levels of CTL against the native peptides from
ND90 PBMC regardless of the peptide used for stimulation.
Donor ND93 CTL raised on OPL2 responded best to native
KLWG-9 peptide with 555 spots per 5 9 104 PBMC, but
curiously not to the 12-mer, as was observed with ND51.
CTL from donor ND94, raised on native KLWG-9 peptide,
responded weakly to KLWG-12 and cognate KLWG-9
peptides compared to other donors. All CTL except ND90
responded strongly to the same peptide they were stimu-
lated with cognate peptide (data not shown).
In view of the fact that KLWG-12 peptide was eluted
from a brain tumor cell line and demonstrated a stronger
Fig. 4 Peptide-HLA class I
interactions a KLWGLTPKV
(KLWG-9).
b KLWGLTPKVTPS (KLWG-
12), c FLWGLTPKV (OPL1),
d FLFGLTPKV (OPL2).
Peptide is shown as a stick
structure. HLA-A*0201 is
shown as a ribbon structure.
Hydrogen bonds between the
peptide and HLA-A*0201 are
shown by dotted lines and
labeled with the residue number
on the HLA molecule
Table 3 Peptide-reactive CTL elicited from normal donors responding to native KLWG-12 and KLWG-9 peptide epitopes as measured by
IFN-c ELISPOT
Donor ‘‘KLWG’’ target CTL raised on: KLWG-9 CTL raised on: FS-OPL1 CTL raised on: FS-OPL2
ND51 12-mer 439 (0.05) 52 (0.18) 19 (0.45)
ND51 9-mer 443 (0.02) 42 (0.21) 7 (0.29)
ND78 12-mer 88 (0.15) 97 (0.03) 25 (0.19)
ND78 9-mer 77 (0.29) 105 (0.03) 15 (0.23)
ND90 12-mer 9 (0.49) 11 (0.14) 8 (0.24)
ND90 9-mer 7 (0.39) 1 (0.39) 5 (0.33)
ND93 12-mer 68 (0.24) 49 (0.29) 27 (0.24)
ND93 9-mer 68 (0.15) 51 (0.16) 555 (0.01)
ND94 12-mer 30 (0.10) 5 (0.26) 0 (0.60)
ND94 9-mer 57 (0.37) 3 (0.36) 0 (0.17)
Table shows the number of spots from a 4th round of stimulation from CTL raised on OPL and tested on wild-type KLWG peptides. CTL were
tested against an irrelevant HIV peptide, and background spots were subtracted from totals. Values listed are IFN-c-secreting CTL from 5 9 104
PBMC per well using KLWG peptide-pulsed T2 cells as targets. Numbers in parentheses indicate coefficient of variation
Cancer Immunol Immunother (2011) 60:1319–1332 1327
123
affinity for HLA-A2 than the 9-mer, it was essential to
determine whether CTL could be generated against the
KLWG-12 peptide (Table 4). Therefore, normal human
PBMC were stimulated weekly with KLWG-12 peptide as
stated earlier. Donor ND117 CTL raised on KLWG-12
responded *35% more strongly to cognate KLWG-12
peptide than to KLWG-9 epitope with 279 and 181 spots,
respectively. Inhibition of CTL recognition of peptide-
loaded MHC class I molecule with W6/32 mAb demon-
strates the specificity and MHC dependency of the
anti-peptide CTL. ND117 CTL raised on KLWG-9 peptide
responded similarly to KLWG-9 and KLWG-12 peptides
with 204 and 209 spots, respectively. Blocking MHC class
I with W6/32 mAb again showed little response with 35
and 47 spots per 5 9 104 PBMC.
CTL cytotoxicity assay
At week 5 of CTL generation, we tested anti-peptide CTL
for the ability to lyse GBM tumor cells in an HLA-A2-
restricted manner in 51Cr release assays. At an effector-to-
target ratio of 100:1, 34% of CRL2610 GBM tumor cells
were lysed by ND 51 anti-KLWG-9 CTL (Fig. 5a). Pep-
tide-pulsed T2 cells were also lysed by ND51 CTL raised
on KLWG-9.
Anti-OPL1 CTL generated from donor ND51 were also
evaluated for the ability to lyse GBM tumor cells. At a
100:1 effector to target ratio, anti-OPL1 CTL lysed 33% of
CRL2610 tumor cells (Fig. 5b). Anti-OPL1 CTL from
ND51 were tested for their ability to lyse T2 cells pulsed
with the 12-mer peptide KLWG-12. At a 100:1 E:T ratio,
anti-OPL1 CTL showed 48% lysis, whereas the anti-
KLWG-9 CTL only showed 37% lysis.
Anti-KLWG-12 CTL generated from ND117 demon-
strated the ability to kill 41% of U-87 glioblastoma tumor
cells at an E:T ratio of 100:1, (Fig. 5c, solid diamonds).
MHC class I–dependent lysis of U-87 was inhibited with
the addition of blocking antibody W6/32 (Fig. 5c, solid
circle). In all cytotoxicity assays, NK activity was between
3 and 5%.
Discussion
Tumors expose their protein contents to CTL by presenting
them as peptides on cell surface MHC class I molecules.
One of the most direct methods for identifying tumor-
associated peptides is to elute them from MHC molecules
on the tumor cell surface and perform tandem mass spec-
trometry on the eluted peptides to determine their
sequences. Because tumor cells are genetically and trans-
criptionally unstable, it is possible that normal transcrip-
tional and translational machinery are dysregulated,
leading to translation of frameshifted and other abnormal
peptides. The abnormal proteins that result should be
ubiquitinylated and targeted to the proteasome where their
subsequent peptides would be sampled by MHC molecules
and presented on the tumor cell surface for T-cell recog-
nition [15–19, 33].
Mass spectrometric analyses of peptides acid-eluted
from GBM tumor cell lines, T98g and CRL2610, resulted
in identification of several potential antigenic peptides. Of
particular interest was a 12-amino acid frameshift peptide
derived from the HSD3B7 gene. It was predicted to have a
very high SYFPEITHI binding score. This peptide was not
found in acid elutions from CRL2610 or PBMC from two
unrelated healthy donors using the same LC–MS/MS
methods. Interestingly, frameshifts from the zinc finger
protein 493 (ZNF493) gene were common to both T98g
and CRL2610, suggesting that this gene may be predis-
posed to transcriptional or translational infidelity. ZNF493
has 3 known variants; isoform 1 lacks 2 exons and utilizes
a downstream start codon, while isoform 2 includes an
exon from a putative 30 untranslated region.
Neither PBMC nor normal brain tissue is available from
the same patient for which the T98g cell line was derived.
However, it is unlikely that this frameshift results from a
unique, patient-specific mutation for 2 reasons. First, PCR
amplification of the RNA region flanking the KLWG
peptide resulted in a splice variant present in 5 GBM
tumors, one primary cell line and all 4 established GBM
cell lines, but not in normal brain tissue (Fig. 2). Second,
Table 4 Peptide-reactive CTL elicited from ND117 responding to native KLWG-12 and KLWG-9 peptide epitopes as measured by IFN-cELISPOT
Donor KLWG ‘‘Target’’ CTL Raised on KLWG-9 CTL Raised on KLWG-12
ND117 12-mer 209 (0.11) 279 (0.09)
ND117 12-mer ? W6/32 35 (0.4) 34 (0.11)
ND117 9-mer 204 (0.11) 181 (0.21)
ND117 9-mer ? W6/32 47 (0.12) 33 (0.12)
Table shows the number of spots from a 4th round of stimulation from CTL generated on KLWG-12, KLWG-9, and tested on wild-type KLWG
peptides with or without MHC class I blocking antibody W6/32. CTL were tested against an irrelevant HIV peptide, and background spots were
subtracted from totals. Values listed are IFN-c-secreting CTL from 5 9 104 PBMC per well using KLWG peptide-pulsed T2 cells as targets.
Numbers in parentheses indicate coefficient of variation
1328 Cancer Immunol Immunother (2011) 60:1319–1332
123
KLWG-12 was identified from T98g eluates, and CTL
raised against the peptide kill CRL2610 and U-87, unre-
lated GBM cell lines. It is clear from Fig. 2 that two splice
variants exist for HSD3B7 in tumor cells and tissue, but not
in normal brain tissue. According to DNA sequence anal-
ysis, the 438-bp PCR product contains exons 2, 3, and 4,
while the 282-bp band contains exons 2 and 4 spliced
together. Unfortunately, we did not find evidence for mis-
splicing in the 282-bp band, even after cloning the PCR
products and sequencing nearly 100 clones. Alternative and
aberrant splicing is well-known in cancer [34–36]. Epi-
dermal growth factor receptor VIII is one well-known
example of a prevalent splice variant in GBM that is also a
target for immunotherapy [37].
It is possible that the junction between exons 2 and 4 is
problematic for translational machinery resulting in ribo-
somal frameshifting, stalling, or slippage [38–40]. These
mechanisms may lead to intermittent expression of the
KLWG peptide such that some transcripts are in frame and
some are translated out of frame. We are currently
addressing this complex question.
The 12-mer peptide contains a 9-mer epitope embedded
in the parent 12-mer that was predicted to bind HLA-
A*0201 by computer algorithms. Interestingly, both the
12-mer and the 9-mer bind to recombinant HLA-A*0201 in
a refolding assay, confirming the hypothesis that KLWG-
12 binds to HLA-A*0201. Surprisingly, the 12-mer bound
to cell surface HLA-A*0201 with a relative affinity three
times stronger than the 9-mer and at least twice as strongly
as OPL1 which contains a preferred phenylalanine (F) at
P1 (Table 2). Although there are no crystal structures
greater than 10 amino acids bound to an HLA molecule,
the molecular modeling scores (higher values indicate
stronger binding, unlike IC50 values) are in agreement with
the cell-based relative affinity measurement, IC50.
Although KLWG-12 demonstrated the strongest relative
affinity to HLA-A*0201, stimulation of normal donor
PBMC with KLWG-9 elicited the strongest responses
among 5 normal donors. CTL were raised on KLWG-9,
KLWG-12, and the two 9-mer OPLs. The rationale for
raising CTL against 9-mers is that functional APCs are
more likely to process and present 9-mers, while tumor
cells may have relaxed antigen presentation mechanisms
such that they present longer peptides. However, the rela-
tive affinity for KLWG-12 peptide for HLA-A2 was 3-fold
stronger than the 9-mer, so CTL were also generated
against KLWG-12. Starting at week five of stimulation,
CTL were assayed by ELISPOT for reactivity only to
native KLWG-12 and KLWG-9 peptides (Table 3).
Responses from different donors to the peptides were
variable, as observed in previous studies [30].
To discuss these findings in a vaccine setting, if donor
ND78 had a glioblastoma expressing KLWG-12 and was
immunized with native KLWG-9, he/she would not
respond as well as if he/she would have been immunized
with OPL1. Similarly, one might argue that donor ND93
should only be immunized with OPL2 because other pep-
tides only sub-optimally stimulated anti-KLWG CTL. CTL
from donor ND51 responded equally well to native
KLWG-12 and KLWG-9, but were marginally stimulated
by OPL1 and only weakly stimulated by OPL2. Although
donor ND90 PBMC were stimulated by a positive control
influenza peptide (data not shown), we were not able to
0
10
20
30
40
50
60
70
80
90
100
100:1 50:1 25:1 12.5:1
% S
peci
fic
Lys
is
E:T Ratio
(A) CTL raised on KLWG-9
(B) CTL raised on OPL1
(C) CTL raised on KLWG -12
Fig. 5 Lysis of target and GBM tumor cells. CTL at indicated E:T
ratios were incubated with 51Cr-labeled target cells (5 9 103 cells/
well), and 51Cr release was measured after 8 h. a ND 51 CTL raised
on native KLWG-9 peptide b ND 51 CTL raised on OPL1 peptide.