Functional and structural characteristics of NY-ESO-1 related HLA-A2
restricted epitopes and the design of a novel immunogenic analogue
Andrew I. Webb†¶, Michelle A. Dunstone†¶, Weisan Chen§, Marie-Isabel Aguilar†,
Qiyuan Chen§, Heather Jackson§, Linus Chang‡*, Lars Kjer-Nielsen‡, Travis Beddoe†,
James McCluskey‡*, Jamie Rossjohn †1 and Anthony W. Purcell‡*1
† The Protein Crystallography Unit and Department of Biochemistry and Molecular Biology,
School of Biomedical Sciences, Monash University, Clayton, Victoria 3168, Australia
§ T cell laboratory, Ludwig Institute for Cancer Research, Austin & Repatriation Medical
Centre, Heidelberg, Victoria 3084, Australia
‡ Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria
3010, Australia.
* ImmunoID, University of Melbourne, Parkville, Victoria 3010, Australia.
¶ Andrew Webb and Michelle Dunstone contributed equally to this work.
Running title: Rational design of tumor antigen analogues
1 Joint senior and corresponding authors. Address all enquiries and reprint requests to either Dr Anthony W. Purcell ([email protected]) Ph: (613)8344991 Fax: (613) 93471540 or Dr Jamie Rossjohn ([email protected]) Ph: (613)9905 3736 Fax: (613) 9905 4699
JBC Papers in Press. Published on March 5, 2004 as Manuscript M314066200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Summary:
Peptide based immunotherapy is one of the current experimental therapeutic regimes for
human malignant disease. NY-ESO-1, a commonly expressed tumor antigen of the Cancer-
Testis family, is expressed by a wide range of tumours but not found in normal adult somatic
tissue, making it an ideal cancer vaccine candidate. Peptides derived from NY-ESO-1 have
shown pre-clinical and clinical trial promise, however biochemical features of these peptide
have complicated their formulation and led to heterogeneous immune responses. We have
taken a rational approach to engineer a HLA A2-restricted NY-ESO-1 derived T cell epitope
with improved formulation and immunogenicity to the wild type peptide. To accomplish this
we have solved the X-ray crystallographic structures of HLA A2 complexed to NY-ESO 157-
165 and two analogues of this peptide in which the C-terminal cysteine residue has been
substituted to Alanine or Serine. Substitution of Cysteine by Serine maintained peptide
conformation yet dramatically reduced complex stability, resulting in poor CTL recognition.
Conversely, substitution with alanine maintained complex stability and CTL recognition.
Based on the structures of the three HLA A2 complexes we incorporated 2-Aminoisobutyric
acid, an iso-stereomer of Cysteine, into the epitope. This analogue is impervious to oxidative
damage, cysteinylation and dimerisation of the peptide epitope upon formulation that is
characteristic of the wild type peptide. Therefore, this approach has yielded a potential new
therapeutic molecule that satiates the hydrophobic F pocket of HLA A2 and exhibited
superior immunogenicity relative to the wild type peptide.
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Introduction:
Class I major histocompatibility complex (MHC) molecules play a crucial role in immune
surveillance by selectively binding to intracellular peptide antigens (Ag) and presenting them
at the cell surface to CD8+ T lymphocytes (TCD8), including cytotoxic T lymphocytes (CTL).
Eradication of tumors is associated with a robust cytotoxic T cell response to antigens
expressed by the tumor (tumor associated antigens (TAA)). Because many TAA are self
proteins or closely related to self proteins they tend to be poorly immunogenic (1-5).
Moreover, many TAA-derived peptides are not strong binders to class I molecules making
strategies that revolve around tumor Ag delivery poor inducers of CD8 T cell immunity (6).
Synthetic peptide-based vaccines offer a flexible, relatively simple and cost-effective way to
treat a variety of human diseases, including the immunotherapy of cancer. Moreover,
synthetic peptides are easily engineered to improve the efficacy of the immunogen. Such
engineering may include optimizing target MHC class I binding by substituting key residues
with more appropriate anchor residues. In addition, peptide-based therapeutics can be
engineered to improve formulation and storage properties and strategies exist to protect labile
peptide bonds by incorporating non-peptidic structures (7-11). Several studies have
incorporated non-natural amino acids in peptidic structures to improve compound stability
and maintain T cell cross-reactivity. For example, some studies have used non-natural amino
acids with modified side chains that approximate the natural amino acid (10,11) or by
modifying peptide bonds by introducing β-amino acids (12,13), reducing peptide bonds from
the natural amine bonds to aminomethylene (14,15) or generation of partially modified retro-
inverso pseudopeptides (8,16,17).
The search for appropriate TAA for vaccination and immunotherapy has extended to several
classes of tumor antigens. Ideally such candidates are expressed solely in cancerous tissue and
are essential for the malignant phenotype, however, few examples of such antigens exist.
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More often TAAs are self proteins over-expressed in tumors or self-proteins that contain
mutations that may or may not be discernable by the immune system. The risk of potential
autoimmune complications in eliciting anti-tumor immunity requires strategies to minimize
autoimmunity. One such strategy is to limit the immune response towards tumor specific
epitopes (e.g. in mutated antigens) or to a few defined and easily monitored epitopes rather
than whole antigen.
Boon and colleagues cloned the first human tumor antigen capable of eliciting spontaneous
CTL responses in melanoma patients (1). This antigen, known as MAGE-A1, is expressed
only in normal testis, yet is frequently found in many different cancers. This expression
pattern has led to MAGE and related antigens being termed cancer-testis antigens. Because
normal testis germ cells do not express class I MHC molecules, this family of antigens has
been extensively studied by the tumor immunotherapy community. NY-ESO-1 is another
cancer testis Ag, expressed in many different types of tumors, including melanoma, breast,
lung and bladder cancers. In addition to its widespread expression by different cancers, it is
also immunogenic in patients with late stage disease, with evidence of spontaneous humoral
and cellular immune responses towards this antigen (18). Both Class I and Class II restricted
T cell determinants have been identified making NY-ESO-1, or peptides derived from it,
potentially useful vaccine components (19-27). Clinical evidence suggests that CTL specific
for NY-ESO-1 determinants can stabilize malignant disease and eradicate metastases. Peptide
vaccination with NY-ESO-1 determinants has been very promising, but along the way these
studies have highlighted problems of stability and bioavailability associated with peptide
immunization and the frequent failure to elicit robust CTL that kill tumors (21,23,28).
Three peptides from an overlapping region of the NY-ESO-1 protein (155-163
QLSLLMWIT, 157-165 SLLMWITQC, and 157-167 SLLMWITQCFL) have previously
been reported as HLA-A*0201–restricted determinants recognized by tumor-reactive TCD8
from a melanoma patient (18). Despite poor binding to HLA-A2, tumor-reactive TCD8 clones
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mainly recognise the NY-ESO157-165 determinant (29). The immunogenicity of these peptides
was first evaluated in a trial vaccination of cancer patients in which a mixture of the peptides
were administered intradermally to patients bearing NY-ESO-1+ tumors (28). A vigorous TCD8
response to NY-ESO157-167 was observed, whereas reactivity against NY-ESO157-165 appeared
later and at a lower level. The TCD8 response to NY-ESO peptide vaccination has also been
examined by HLA-A2/peptide tetramer analysis and revealed a heterogeneous response
directed against several distinct overlapping epitopes, including cryptic determinants
generated by aminopeptidase activity (24). Thus, only CTL recognizing the precise NY-
ESO157-165 determinant also recognize the endogenously processed determinant on NY-ESO+
tumor cells, probably because it is the only constitutively presented determinant on tumor
cells (20).
Analogs of NY-ESO157-165 where the C-terminal Cys residue has been replaced with more
conventional anchor residues, namely Leu9 and Val9 analogs have been generated(25).
Whilst these analogs bind more efficiently to HLA-A2 and are recognised by CTL raised
against the natural NY-ESO157-165 peptide, they do not induce effective anti-tumor CTL in
vivo. Indeed, the presence of the Cys at the C-terminus seems critical for generating CTL that
recognise endogenously processed NY-ESO determinants on tumor cells. The presence of this
amino acid causes problems with formulation due to oxidative damage and dimerisation, both
of which reduce the efficacy of the peptide Ag as an immunogen (25). In this study we have
investigated the structure of NY-ESO157-165 complexed to HLA A*0201 and compared it to
the C9A and C9S structures which are more easily formulated and potential vaccine
candidates (see Table 1). We have also examined the functional recognition of these
analogues using a CD8+ T lymphocyte lines derived from melanoma patients immunized
with overlapping peptides spanning NY-ESO 155-167 (24) that respond to NY-ESO157-165. In
our studies we have been careful to pre-treat all the peptides including the Cys containing
peptides with a reductant to prevent dimerisation or cysteinylation of the peptides which
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could mask the recognition of the wild type peptide relative to the analogs. This allowed for
the first time a systematic analysis of relative antigenicity of the wild type peptide and
analogues. Finally we use structure guided design to test an analog that should satisfy the
Cys-requirement of anti-tumor CTL by substituting the Cys 9 for a non-natural isosteric
analog of this residue 2-amino-isobutyric acid (Abu).
Experimental procedures
Peptides
All peptides were synthesised using standard Fmoc synthesis and synthesised by Auspep Pty
Ltd (North Melbourne, Victoria, Australia). All peptides were purified to >85% purity by
preparative RP-HPLC and purity determined by LC-MS using an Agilent 1100 LC-MSD SL
ion trap instrument and a Stable Bond RP C18 column (100x0.5mM i.d. column) (see Table
1). Peptides were dissolved in DMSO to a final concentration of 10-100mg/ml.
Expression, purification, crystallization and structure determination
Truncated HLA A*0201 class I heavy chain, encompassing residues 1-274 were expressed as
inclusion bodies (30) using the BL21 (RIL) strain of E. coli. At an A600 of 0.6, cultures were
induced with 1mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 12 hours, bacteria
were lysed in 50mM Tris-HCl pH 8.0, 1% TritonX-100, 1% Sodium deoxycholate, 100mM
NaCl and 10mM DTT. Inclusion bodies were isolated by centrifugation after washing with
50mM Tris-HCl, 0.5% TritonX-100, 100mM NaCl, 1mM NaEDTA, 1mM DTT, pH 8.0, and
washing in 50mM Tris-HCl, 1mM NaEDTA, 1mM DTT, pH 8.0, and then solubilized in
50mM Tris, 8M Urea, 10mM NaEDTA, pH 8.0 with the protease inhibitors 1µg/ml Pepstatin
A and 200µM phenylmethylsulfonyl fluoride (PMSF). Recombinant protein (30mg A2 heavy
chain and 10mg β2m) was refolded with 6mg of peptide reconstituted in 3M guanidine-HCl,
10mM NaAcetate, and 10mM NaEDTA, pH 4.2, in a refolding buffer composed of 0.1M Tris,
2mM EDTA, 400mM L-Arginine-HCl, 0.5mM Oxidized Glutathione, 5mM Reduced
Glutathione pH 8.0 at 4°C for 72 hours. Following refolding, protein was dialyzed overnight
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against Milli Q using a 6-8,000 kDa MWCO dialysis membrane (Spectrum, California, USA).
Protein was concentrated by ion exchange on a DE52 column (Whatman, Maidstone, Kent,
U.K.), and subsequently purified by size exclusion on a Superdex 75pg gel filtration column
(Amersham Pharmacia Biotech, Uppsala, Sweden), and a final ion exchange on a MonoQ HR
10/10 column (Amersham Pharmacia Biotech). Quantitative analysis was based on
comparisons to BSA protein standards using SDS-polyacrylamide gel electrophoresis.
Protein was concentrated to 10mg/ml for use in crystallization trials.
Crystallization
Large cubic crystals (0.3 x 0.3 x 0.3mm) were obtained using the hanging drop vapour
diffusion technique at room temperature. The crystals were grown within 3-5 days by mixing
equal volumes of 10mg/ml HLA A2-NY-ESO-1 peptide (and analogues thereof) with the
reservoir buffer (2.0M Ammonium sulfate, 0.1M Na citrate, pH 6.5). The crystals belong to
space group P213 with unit cell dimensions a=b=c 117.90Å, α=β=γ = 90°. The crystals
were flash frozen prior to data collection using crystals that had been soaked in 15% glycerol.
One 2.2Å and two 2.5Å data sets were collected for the NY-ESO-1 series and scaled using the
HKL suite (31). For a summary of statistics see Table 1.
Structure determination
The structure was solved by the molecular replacement method, using the program AmoRe
within the CCP4 Suite (32). The previously solved monomeric HLA A2 structure (PDB code:
1DUY) (33), minus the peptide, was used as the search probe. A clear peak in the rotation
function yielded one clear solution in the translation function that packed well within the unit
cell. Following rigid body fitting in AmoRe the molecular replacement solution had an Rfac
and correlation coefficient of 68.2 and 38.1 respectively. Unbiased features in the initial
electron density map, including that of the NY-ESO-1 peptide confirmed the correctness of
the molecular replacement solution. The progress of refinement was monitored by the Rfree
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value (4% of the data) with neither a sigma, nor a low resolution cut off being applied to the
data. The structure was refined using rigid-body fitting of the individual domains followed by
the simulated-annealing protocol implemented in CNS (version 1.0) (34), interspersed with
rounds of model building using the program 'O' (35). Tightly-restrained individual B-factor
refinement was employed, and bulk solvent corrections were applied to the data set. Water
molecules were included in the model if they were within hydrogen-bonding distance to
chemically reasonable groups, appeared in Fo - Fc maps contoured at 3.5σ, and had a B-factor
less than 60 Å2. See Table 1 for summary of refinement statistics and model quality.
HLA A*0201 assembly assay
The cDNA encoding the ectodomain of HLA class I molecules HLA A*0201 (amino acids 1-
276) were inserted into pET30 (Novagen) vector and verified by DNA sequencing. Inclusion
body protein of the hc and β2m were prepared as described (30,36,37). In vitro assembly of
HLA A2-peptide complexes in micro-assembly reactions was initiated by sequential addition
of recombinant β2m (2µM) and HLA A2 hc (3µM) to peptide (30µM) in a buffer containing
100 mM Tris pH 8.0, 0.4M arginine, 0.5 mM oxidised glutathione, 5 mM reduced
glutathione, 2 mM EDTA, 0.2 mM PMSF in a final volume of 1 ml. The assembly reaction
mixture was allowed to proceed at 4oC for 48h and aggregated material removed by
centrifugation. Quantitation of assembled HLA class I complexes was performed by capture
ELISA; briefly 96-well plates were coated with affinity-purified pan class I specific
monoclonal antibody W6/32 at 10µg/ml, washed 3 times with PBS containing 0.05% Tween-
20 (PBST), and blocked with PBST containing 1% BSA. Properly assembled and correctly
conformed HLA-peptide complexes were captured and detected by incubation with HRP-
conjugated rabbit anti-human β2m polyclonal antibodies (DakoCytomation, Glostrup,
Denmark A/S) and the chromogen O-phenylene diamine (OPD, Sigma).
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Thermostability measurements of recombinant class I complexes using circular
dichroism
Circular dichroism (CD) spectra were measured on a Jasco 810 spectropolarimeter using a
thermostatically controlled cuvette at temperatures between 20-90°C. Far-UV spectra from
195 nm to 250 nm were collected with a five seconds/point signal averaging and were the
accumulation of ten individual scans; 218 measurements for the thermal melting experiments
were made at temperature intervals of 0.1°C at a rate of 1°C/min. The midpoint of thermal
denaturation (Tm) for each protein was calculated by taking the first derivative of the
elipticity data and identifying the inflexion point, which represents the Tm for each protein.
All complexes were measured at 20µg/ml in a solution of 10mM Tris, 150mM NaCl, pH 8.0.
T cell lines and Interferon-γγγγ assay
The NY-ESO-1 specific CTL lines with specificity against NY-ESO-1157-165 were derived
from DTH biopsy after HLA A2+ patients bearing an NY-ESO+ tumors received NY-ESO-1
peptide157-165 peptide vaccination. This clinical trial was conducted at the Ludwig Institute for
Cancer Research at the Austin Hospital in Melbourne, Australia. It was approved by the
Human Research Ethics Committee of Austin Health and the patient provided written
informed consent. Due to potential oxidation of the wild type peptide and the rapid
cysteinylation of this peptide in tissue culture medium during Ag presentation assays, all
peptides were treated with 500 µM of Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP)
(Pierce Endogen, IL, USA) which reduces oxidation, dimerisation and other modification of
the Cysteine residues without affecting T cell reactivity, allowing accurate comparison of T
cell cross reactivity (38). Transporter associated with antigen processing (TAP)-deficient T2
cells were pulsed with graded concentrations of the peptides at room temperature for 45mins
and then washed. T cells were then added along with Brefeldin A (BFA) at final concentration
of 10 µg/mL. The cells were incubated for a further 4 hours, harvested and stained with anti-
CD8-Cychrome conjugate in 50µl of PBS at 4oC for 30min, washed and fixed with 1%
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paraformaldehyde. The cells were permeabilised with 0.2% Saponin and intracellular IFN-γ
that had accumulated in the presence of BFA was detected using an anti-IFN-γ-FITC
conjugate. 100,000 events were acquired on a FACScalibur flow cytometer and the data
analysed with Flowjo software (TreeStar, San Carlos, CA).
Results
Structure of NY-ESO-1 157-165 peptide complexed to HLA A2
The HLA A2- NY-ESO complex, and analogues thereof, have been crystallized in the cubic
space group P213, with one molecule per asymmetric unit, and diffracted to a resolution of
2.5Å or better The structures were determined via molecular replacement, using a previously
determined HLA A2 structure as the search probe (1DUY (39)). The structure of HLA-
A*0201 complexed to the wild type NY-ESO157-165 peptide has been refined to 2.2 Å to an
Rfac and Rfree of 22.8 and 26.7% respectively; the structure of the C9A analogue has been
refined to 2.3 Å to an Rfac and Rfree of 23.6 and 27.3% respectively; the structure of the C9S
analogue has been refined to 2.5 Å to an Rfac and Rfree of 23.0 and 27.9% respectively (See
Table 2 for a summary of the refinement statistics for each analogue). The three structures
comprise residues 1-274 of the HLA A2 heavy chain, residues 1-99 of β2-microglobulin, and
nine residues of the bound peptide, one sulfate ion and a variable number of water molecules.
The electron density for the bound NY-ESO peptide, and the two analogues (Fig 1a-d), as
well as the interacting residues was unambiguous. The structure of the NY-ESO-1157-165
complex, the highest resolution complex, will be discussed initially, followed by the salient
aspects of the analogue structures. The overall structure of the HLA A2 complex was very
similar to those reported previously (e.g. (30,40-46). Thus our analysis focuses on the peptide
conformation and cleft interactions of the NY-ESO peptides bound to HLA-A2. The NY-
ESO-1157-165 peptide is bound in an extended conformation, containing a centrally-located
bulge at P4-Met and P5-Trp, two prominent, surface exposed hydrophobic residues (Fig1a).
These two residues, along with the upward-pointing side chains of P7-Thr and P8-Gln are
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likely to contact the TcR. The NY-ESO peptide is bound between the helical jaws of the
antigen-presenting domain (Fig 1a) making considerable polar contacts with the HLA A2
molecule along the length of the peptide (Table 3, Fig 2), with 12 hydrogen bonds and 12
water-mediated hydrogen bonds, as well as a number of van der Waals interactions.
The average temperature factor for the bound peptide is 34Å2, whereas the increased mobility
of the P4-Met (B-factor 45Å2) and P5-Trp (43Å2) is consistent with the limited number of
contacts these residues make with the HLA A2 heavy chain (Table 3). The buried and anchor
residues at positions P2, P3, P6 and P9 are unlikely to interact with the TcR. Conversely, P1-
Ser is solvent exposed and also a potential TcR contact residue. The peptide residues P4-Met,
P5-Trp, P7-Thr and P8-Gln, have previously been implicated in T cell recognition by an
alanine scan of the NY-ESO157-165 peptide using tumor-reactive CTL lines (22). The P9-Cys
residue is buried and participates in anchoring interactions with the hydrophobic F pocket.
The N-terminal P1-Ser is strongly tethered within the cleft, with the main chain forming
hydrogen bonds with the side chains of Tyr 7, Tyr 159 and Tyr 171, whilst the side chain
stacks against Trp 167, and the P1-Seroγ group forming a H-bond with Glu 63. Glu 63 also
forms a H-bond with the main chain of P2-Leu, a hydrophobic anchor residue that
correspondingly sits in the hydrophobic B pocket, comprising Tyr 7, Phe 9, Met 45, Val 67
and Tyr 99 of the A2 heavy chain. Tyr 99 also interacts with the P3-Leu sidechain, a residue
that also sits in a hydrophobic pocket. An abrupt alteration in the main chain conformation at
P3-Leu (Φ=-65,Ψ=154), P4-Met (Φ=-73,Ψ=-18) results in the observed bulged conformation
of the bound peptide. Residues in this region of the peptide ligand form limited side chain or
backbone contacts with the HLA A2 heavy chain residues.
The hydrophobic P6-Ile side chain sits within a polar pocket of HLA A2, forming van der
Waals contacts with Arg 97, although it’s guanadinium group is orientated away from this
pocket, forming a salt bridge with Asp 77, a residue located in the F-pocket (Fig 3). In
comparison to some other HLA A2 structures the positioning of Arg 97 is varied such that in
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a previously determined A2 complex (PDB code 1DUY), Arg 97 does not form a salt bridge
with Asp 77. Instead Arg 97 points “upwards” towards the bulged section of the bound
peptide. Arg 97 is sandwiched between Tyr 99 and Tyr 116, with Tyr 116 being orientated
towards the D pocket. In our NY-ESO157-165 complex, Tyr 116 is orientated towards the F-
pocket. Thus, the positioning of Arg 97 also impacts significantly on the positioning of Tyr
116, a key F pocket residue.
The side chains of P7-Thr and P8-Gln also interact with the heavy chain, with water-mediated
contacts predominating for the residue at P7, whilst Gln 8 also forms van der Waals
interactions with Thr 73 and Val 76. The anchor residue at position 9 is unusual in that it is
occupied by a Cys residue. The main chain is tethered by a number of H-bonds to Asp77, Thr
143, Lys 146 as well as forming some water-mediated H-bonds. The side chain sits within
the pocket, making vdw contacts with the polar side chains, Asp 77 and Thr 143. The Cα and
Cβ group forms van der Waals interactions with Trp 147, whereas the sulfur moiety of the
P9-Cys is neither in the correct geometry nor within suitable hydrogen-bonding distance to
make H-bond contacts with F pocket residues. Instead, the sulfur moiety exclusively forms
vdw contacts with Thr 143, Leu 81 and Asp 77.
Structures of C-terminally modified analogs of NY-ESO 157-165
The structures the C9A and C9S peptide analogues bound to HLA A2 are extremely similar to
the wild type NY-ESO157-165-HLA A2 complex. Comparison of the wild type to the C9A-
HLA A2 complex yielded an r.m.s.d. 0.10Å for the 383 Cα atoms. Comparison of the wild
type to the C9S-HLA A2 complex yielded an r.m.s.d. of 0.15Å for the 383 Cα atoms.
Variation in the F pocket interactions are largely confined to the terminal functional group of
each residue (R-CH3, R-CH2OH, R- CH2SH). The methyl functionality of P9-Ala is in a
similar position to the Cβ of P9-Ser and P9-Cys. Additional alterations occur to accommodate
the more polar Ser functionality, with the P9-Seroγ makes a direct H-bond to Asp 77 resulting
in small movement of the hydroxyl group relative to the thiol group of P9-Cys. As discussed
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below these subtle changes in F pocket binding lead to substantial changes in complex
stability, suggesting the thiol group of the wild type peptide contributes further stabilizing
influences.
Rational design of a peptidomimetic
Based on the observation that the Cysteine residue and closely related homologous
substitutions (i.e. Ser and Ala) shared very similar structures and that the thiol of the Cysteine
was primarily involved in van de Waals interactions, we substituted the Cysteine for 2-
Amino-isobutyric acid (Abu) a non-natural amino acid that is isosteric for Cysteine. We
anticipated that the replacement of the thiol group with a methyl group would satisfy any
stereochemical anchoring requirement and that indeed the more hydrophobic nature of this
analog may be better suited to anchoring in the hydrophobic HLA A2 F pocket (41) (see
Table 1). This analog was synthesised using standard Fmoc chemistry and unlike the wild
type peptide did not form dimers or become oxidized during synthesis, purification and
storage (data not shown).
Assembly and stability of NY-ESO157-165 and analogues complexed to HLA A2
We used a newly developed HLA A2 assembly assay (37) to assess the binding of the wild
type peptide and each analogue, including the C9Abu analogue, to HLA A2. This assay does
not rely on cell surface stabilization of antibody determinants, but rather utilizes an in vitro
assembly reaction with quantitation by capture ELISA (37,47). As such this assay is less
influenced by cell culture mediated oxidation and modification of Cysteine containing
peptides. Over a peptide concentration range of 0.5 to 10µM each peptide drove assembly of
HLA A2, with wild type and C9A mediating roughly equivalent assembly, C9V slightly
better and C9Abu and C9S slightly worse than wild type (see Fig 4). In order to further
investigate the ability of these analogues to bind to and stabilize HLA A2 we also examined
the thermostability of complexes formed by each analogue with HLA A2 by circular
dichroism (CD). All complexes gave similar spectra at 20°C, however, the mid point thermal
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denaturation revealed compelling differences in the stability of these complexes (Fig 5). C9V
was 4.5°C more stable than the wild type, whilst C9A was of similar stability to the wild type
peptide, with the new C9Abu analogue displaying modest improvement in thermostability of
1.5°C. The C9S analog however was 10°C less stable. The thermostability of complexes is
related to the dissociation constant for the complexes (48) and the half-life of these complexes
on the cell surface (49) and thus will impact on their immunogenicity.
Recognition of analogues by a CD8+ NY-ESO157-165 specific T cell lines
We next examined the ability of two independently derived T cell lines expanded in vitro by
wildtype NY-ESO157-165 pulsed APC to recognise each variant peptide. In order to rule out the
effects of modification of the Cysteine of the wildtype NY-ESO157-165 peptide all experiments
were carried out in the presence of 500µM TCEP, at this concentration of reductant no
dimerisation is observed in vitro (data not shown) and oxidation and cysteinylation is reduced
without affecting T cell function or viability (38). As shown in Figure 6A and B, the C9Abu
was recognised by T cells significantly better than the wild type peptide and other analogues
for the two independent T cell lines derived from patients HH and M121. Moreover, C9Abu
was able to expand cross-reactive CD8+ NY-ESO157-165 specific T cells from PBMCs derived
from immunized HLA A2+ patients (data not shown). A general pattern of reactivity was
observed for both T cell lines such that C9Abu>C9A, C9V>wildtype>C9S>C9L, which did
not correlate directly with binding or stability of the complexes.
Discussion
The structures of HLA A2 complexed to NY-ESO 157-165 and two C-terminally substituted
analogue peptides have been solved to 2.5Å resolution or better. Cysteine is an unusual
anchor residue for a HLA A2 ligand, and prior to this study the exact role of the Cys residue
and in particular the potentially reactive thiol in providing anchor contacts with the
hydrophobic F pocket of HLA A2 was unknown. In fact the majority of HLA A2 structures
encompass complexes in which the peptide ligand terminates in Valine or Leucine. One
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structure with an Alanine terminating peptide (a P9 Leucine to Alanine substituted influenza
virus matrix peptide GILGFVTFA) has been reported previously (44). The conformation of
the HLA A2 heavy chain was conserved in all structures and only small differences in peptide
conformation were observed. A feature of each structure is the presence of a non-canonical P9
anchor residue that only partially satiates the hydrophobic HLA A2 F pocket. Despite their
conserved structure, the analogues demonstrated remarkable differences in stability and
functional recognition by two T cell lines derived from peptide vaccinated patients.
In this study we were able to compare the relative immunogenicity of each analogue to the
wild type peptide by minimizing oxidative damage of the peptide or cysteinylation of the P9-
Cys residue by performing binding and stability assays in vitro and by treating the peptides
with TCEP during Ag presentation assays. This was performed with two independent T cell
lines (from patients HH (Fig 6a) and M121 (Fig 6b). The C9Abu analogue was consistently
recognised more efficiently by the to T cell lines, and as a general rule the following
reactivity pattern was observed C9Abu>C9A, C9V>wildtype>C9S>C9L. This did not simply
correlate directly with binding or stability of the complexes as may be expected (6,50-57), and
is consistent with several other studies that show the immunogenicity of some T cell
determinants is influenced by additional factors (9,58-62).
C9S bound to HLA A2 slightly less efficiently than the wild type peptide, yet demonstrated
drastically worse stabilization of the complexes. C9A and wild type bind and stabilize HLA
A2 equally efficiently suggesting this analogue is equivalent to the wild type peptide in cross-
sensitizing target cells for recognition. The C9V peptide exhibits superior binding and
stabilization of HLA A2; the equivalent functional recognition of this determinant reflects
somewhat diminished recognition on a mole for mole basis given this peptide will generate a
higher determinant density. C9Abu binds more weakly to HLA A2 than the wild type peptide
yet complexes of the two peptides with HLA A2 exhibit equivalent thermostability. Thus,
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although the Abu analogue is not the most stable or the best binder to HLA A2 it still
demonstrates superior immunogenicity to the wild type and other analogues.
Based on the frequency of codons encoding for Cysteine in the human genome, we have
estimated that 14% of T cell epitopes potentially contain Cys residues (38), suggesting that
immune responses to such antigens may frequently be masked by oxidation and
cysteinylation. Moreover, the seminal observations made by Meadows and colleagues (63),
that a peptide originating from SMCY was only recognised by T cells following post-
translational modification of a cysteine residue that involved attachment of a second cysteine
residue via a disulfide bond, highlight the importance of these types of reactions in immunity.
Subsequent studies have indicated this type of modification has profound effects on T cell
recognition (38) and that cysteine modification occurs in a number of different class I MHC-
associated peptides including the epitope reported here. These observations support the notion
that this form of modification has general importance as mechanism of generating
immunogenic T cell determinants. Finally, our strategy of substituting Abu for Cys in T cell
epitopes may have general application, particularly for Cys-terminating epitopes (such as
LCMV glycoprotein determinants in C57BL/6 mice (64)).
It is a standard approach to engineer anchor residues to improve MHC binding characteristics
in epitope based vaccine strategies (6). Whilst this frequently imparts improved MHC binding
it does not always equate to improved immunity towards the naturally processed peptide in
vivo. For example, our data clearly shows that substitution for more appropriate P9 anchor
residues for HLA A2 such as Valine or Leucine, whilst enhancing binding do not increase T
cell recognition and in the case of C9L this substitution is detrimental for T cell recognition
(Fig 6). Interestingly, substitution of Cys with Serine substantially effects complex stability
and T cell recognition, which we hypothesize is due to the large reduction in complex
stability. Given the close nature of these residues and the frequency with which Cys is
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substituted for by Ser in homologous substitution experiments this highlights the requirement
for more rational approaches for epitope engineering.
Because TAA are frequently related to self proteins, the available T cell repertoire may be
diminished due to thymic and peripheral deletion of those clonotypes specific to the very
immunogenic peptides with strong binding ability. As a result many immunogenic tumor
epitopes are relatively poor binders to their cognate class I molecule. Thus, many tumor
epitopes have been engineered to produce heteroclitic responses, as a result of improved
MHC binding. Recent examples include substitution of subdominant anchor residues in an
epitope in a B16 melanoma model (65) and identification of a HER-2/neu heteroclitic epitope
that provides superior protection in mouse model of breast carcinoma (66). The latter adopted
a common strategy of selecting improved epitopes via an alanine scan of the wild type epitope
(58,67). A systematic study by Tangri et al. demonstrated the potential for heteroclitic
epitopes in inducing high avidity cross-reactive anti-tumor CTL against tolerant or weakly
immunogenic TAA (68) based on conservative or semi-conservative natural amino acid
substitutions. As such, this report is one of few studies to successfully incorporate non-natural
amino acids into T cell epitopes (9-11,69) and highlights the path ahead for rational vaccine
design.
Acknowledgements
A.W.P. is a C.R. Roper Fellow of the Faculty of Medicine, Dentistry and Health Science at
the University of Melbourne. J.R. and W.C. are supported by Wellcome Trust Senior
Research Fellowships in Biomedical Science in Australia. This work was supported by the
NH&MRC, the Roche Organ Transplantation Research Foundation and the Juvenile Diabetes
Research Foundation. We thank the staff at BioCARS and the Australian Synchrotron
Research Program for assistance.
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Co-ordinates of the HLA A2- NY-ESO157-165, C9A and C9S complexes have been deposited
in the PDB databank accession numbers 1S9W, 1S9X and 1S9Y respectively.
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Figure Legends:
Figure 1: Structures of HLA A2 complexed to NY-ESO 157-165 and analogues A) HLA
A*0201/ESO157-165 complex 2.2Å electron density omit map with a cut-away view of the
peptide bound to the HLA-A2 Ag binding cleft. The same view is presented for the 2.5Å C9S
complex structure (B) and the 2.3Å C9A complex structure (C). Very similar conformations
were observed for all complexes, highlighting the exposed Met-4, Trp-5, Thr-7, Gln-8
residues. (D) View of the wildtype NY-ESO157-165 peptide in the cleft of HLA A2 as seen
from above.
Figure 2: Image of the cleft contacts made between the HLA A2 heavy chain and the NY-
ESO 157-165 peptide with H-bond contacts only shown. Numerous H-bond and van der
Waals contacts exist between the peptide and the HLA A2 cleft residues, including anchoring
interactions between P2-Leu and B pocket residues and P9-Cys and F pocket residues. These
interactions are summarized in Table 3. A large number of peptide-main chain H-bond
interactions were observed for this complex relative to other HLA A2 complexes (45,46)
which tend to have more water mediated H bonding networks.
Figure 3: Differences in peptide conformation are mainly restricted to the terminal functional
groups of the P9 amino acid. Detailed view of the F-pocket interactions between the C-
terminal Cysteine 9, Serine 9 and Alanine 9 of the wild type, C9S and C9A analogues of the
NY-ESO 157-165 peptide.
Figure 4: Assembly of HLA-A2 with peptide analogues as revealed by capture ELISA of in
vitro assembled complexes formed at different peptide concentrations. Capture of
conformationally sound HLA A2-peptide complexes was quantitated by capture with W6/32
and readout with a HRP-conjugated anti-β-2-microglobulin monoclonal antibody following
color development at 492nm (as described elsewhere (37)).
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Figure 5: Thermostability of purified HLA A2 complexes formed with NY-ESO 157-165 and
analogues, as revealed by circular dichroism spectropolarimetry. Changes in complex
structure were monitored at θ218nm as temperature was ramped up from 20-90°C and the
fraction unfolded material expressed as a function of temperature.
Figure 6: Recognition of NY-ESO157-165 and analogues by HLA-A2 restricted NY-ESO157-165
specific TCD8 isolated from a peptide vaccinated melanoma patients. T2 cells were pulsed with
graded concentrations of each peptide and T cell response is shown as the percentage of
CD8+ T cells producing IFN-γ. Data is shown for two representative T cell lines from patients
HH (A) and M121 (B).
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Table 1: Peptides used in this study
Sequence Structure at C-
terminus
mass Purity A2
binding
Tm
(°°°°C)
NY-ESO
157-165
SLLMWITQC
NH2
O
SH
1093.5 >95% +++ 57
C9S SLLMWITQS
NH2
OH
O 1077.6 >95% ++ 47
C9A SLLMWITQA
NH2
OCH3
1061.6 >95% ++++ 57
C9L SLLMWITQL
NH2
O
CH3
CH3
1103.6 >95% ND ND
C9V SLLMWITQV
NH2
CH3
OCH3 1089.6 >95% +++ 61.5
C9Abu SLLMWITQAbu
NH2
O
CH3
1075.6 >85% +++ 58.5
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Table 2 Data collection statisticsCrystal NY-ESO C9S C9A X-ray source RU3-HBR APS - Biocars RU3-HBR Detector R-Axis IV++ Q4 R-Axis IV++
Space Group Cell dimensions(Å) (a=b=c)Resolution (Å)
P213117.712.20
P213117.922.50
P213117.952.30
Total No. observations 157846 39141 54324 No. unique observations 27826 18211 22859 Multiplicity 5.7 2.1 2.4 Data completeness (%) 99.9 (100.0) 95.0 (95.5) 92.9 (74.0) I/σI 32.7 (3.4) 13.1 (2.5) 10.5 (2.8) Rmerge
1 (%) 6.1 (42.4) 7.2 (41.7) 5.3 (28.8)
Refinement statistics Crystal NY-ESO C9S C9A Non hydrogen atoms
Protein Water sulfate
31501921
31501601
31491531
Resolution (Å) 50 – 2.2 50 - 2.5 50 – 2.3 Rfactor
2(%) 22.8 23.0 23.6 Rfree
3 (%) 26.7 27.9 27.3 Rms deviations from ideality Bond lengths (Å) Bond angles (°)Impropers (°)Dihedrals (°)
0.0061.230.6924.63
0.0091.380.8425.02
0.0071.260.7024.94
Ramachandran plot Most favoured And allowed region (%)
88.511.2
88.511.2
87.312.4
B-factors (Å2)Average main chain Average side chain Average water molecule r.m.s. deviation bonded Bs
41.743.244.31.44
42.944.138.01.53
48.8149.6747.931.31
Footnote
The values in parentheses are for the highest resolution bin (approximate interval 0.1Å) 1 Rmerge = Σ |Ihkl - <Ihkl>| / ΣIhkl 2 Rfactor = Σhkl | |Fo| - |Fc| | / Σhkl |Fo| for all data except for 4% which was used for the 3Rfree calculation
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Table 3. Interactions between NY-ESO peptide and HLA-A2Peptide HLA-A2 Type of interaction
Ser 1 Trp 167 vdw
Ser 1 Oγ Glu 63 Oε1 H-bondSer1 O Tyr 159 Oη H-bondSer 1 N Tyr 171 Oη
Tyr 7 OηH-bondH-bond
Leu 2 Tyr 7, Phe 9, Met 45, Val 67, Tyr 99
vdw
Leu 2 N Glu63 Oε1 H-bond
Leu 3 Tyr 99, Gln 155, Leu 156, Tyr 159
vdw
Leu 3 N Tyr99 Oη H-bond
Met 4 Lys 66 vdw
Trp 5Gln 155 vdw
Trp 5 O Gln 155 Nε2 H-bond
Ile 6His 70, Thr 73, Arg 97 vdw
Ile 6 N Wat 48 Mediates H-bond to Thr 73 Oγ1, Ala 69 O
Ile 6 O Thr 73 Oγ1 H-bond
Thr 7 Val 152 vdw
Thr 7 N Wat 52 Mediates H-bond to Gln 155 Oε1
Thr 7 Oγ1 Wat 44 Mediates H-bond to Gln 155 Oε1
Thr 7 O Wat 19, Wat 110
Mediates H-bond to Asp77 Oδ1, Arg 97 Nη2
Mediates H-bond to Arg 97 Nη1
Gln 8 Thr 73, Val 76 vdw Gln 8 NE2 Wat 54 Mediates H-bond to Thr 73 Oγ1
Gln 8 O Trp 147 Nε1 H-bond
Cys 9 Asp 77, Thr 80, Leu 81, Thr 143, Trp 147
vdw
Cys Sγ Asp 77, Thr 80, Leu 81 vdw Cys 9 N Asp 77 Oδ1
Wat 65 H-bondMediates H-bond to Asp77 Oδ1, Thr 80 Oγ1
Cys 0 Thr 143 Oγ1 H-bondCys 9 OXT Lys 146 Nζ
Wat 65 H-bondMediates H-bond to Asp77 Oδ1, Thr 80 Oγ1
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Webb et al. Figure 1
Wild type C9S
Wild typeC9A
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Webb et al. Figure 2
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Wild type C9S C9A
Webb et al. Figure 3
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Webb et al. Figure 4
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
Wildtype C9A C9V C9Abu C9Speptides
Ref
olde
d H
LA A
2 (A
492n
m)
0.5uM 1uM 2uM 5uM 10uM
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% U
nfol
ded
Webb et al. Figure 5
0
20
40
60
80
100
35 40 45 50 55 60 65 70 75
WildtypeTm=57°C
C9AbuTm=58.5°C
Temperature (°C)
C9ATm=57°C
C9STm=47°C
C9VTm=61.5°C by guest on February 5, 2020
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Webb et al. Figure 6
0
5
10
15
20
1 x10-(11) 1 x10(-10) 1 x10-(9) 1 x10-(8) 1 x10-(7) 1 x10-(6)
peptide concentration (M)
% A
ntig
en s
peci
fic T
cel
l
WildtypeC9AC9LC9VC9SC9Abu
0
10
20
30
40
2.5 x10-(9) 2.5 x10-(8) 2.5 x10-(7) 2.5 x10-(6)
Peptide concentration (M)
% A
ntig
en s
peci
fic T
cel
l
A
B
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Rossjohn and Anthony W. PurcellJamieHeather Jackson, Linus Chang, Lars Kjer-Nielsen, Travis Beddoe, James McCluskey,
Andrew I. Webb, Michelle A. Dunstone, Weisan Chen, Marie-Isabel Aguilar, Qiyuan Chen,epitopes and the design of a novel immunogenic analogue
Functional and structural characteristics of NY-ESO-1 related HLA-A2 restricted
published online March 5, 2004J. Biol. Chem.
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