Functional and structural characteristics of NY-ESO-1 ... · vaccination with NY-ESO-1 determinants has been very promising, but along the way these studies have highlighted problems

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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References

1. Boon, T., and Vanderbruggen, P. (1996) J. Exp. Med. 183, 725-729

2. Platsoucas, C. D., Fincke, J. E., Pappas, J., Jung, W. J., Heckel, M., Schwarting, R., Magira, E., Monos, D., Freedman, R. S., Engelhard, V. H., Bullock, T. N., Colella, T. A., Sheasley, S. L., Mullins, D. W., Kourilsky, P., and Fazilleau, N. (2003) Anticancer Res 23, 1969-1996

3. Jager, D., Jager, E., and Knuth, A. (2001) J Clin Pathol 54, 669-674

4. Engelhard, V. H., Bullock, T. N., Colella, T. A., Sheasley, S. L., and Mullins, D. W. (2002) Immunol Rev 188, 136-146

5. Kourilsky, P., and Fazilleau, N. (2001) Int Rev Immunol 20, 575-591

6. Sette, A., and Fikes, J. (2003) Curr Opin Immunol 15, 461-470

7. Purcell, A. W., Zeng, W., Mifsud, N. A., Ely, L. K., Macdonald, W. A., and Jackson, D. C. (2003) J Pept Sci 9, 255-281

8. Guichard, G., Connan, F., Graff, R., Ostankovitch, M., Muller, S., Guillet, J. G., Choppin, J., and Briand, J. P. (1996) J Med Chem 39, 2030-2039

9. Guichard, G., Zerbib, A., Le Gal, F. A., Hoebeke, J., Connan, F., Choppin, J., Briand, J. P., and Guillet, J. G. (2000) J Med Chem 43, 3803-3808.

10. Krebs, S., Folkers, G., and Rognan, D. (1998) J Pept Sci 4, 378-388

11. Krebs, S., Lamas, J. R., Poenaru, S., Folkers, G., de Castro, J. A., Seebach, D., and Rognan, D. (1998) J Biol Chem 273, 19072-19079

12. Reinelt, S., Marti, M., Dedier, S., Reitinger, T., Folkers, G., de Castro, J. A., and Rognan, D. (2001) J Biol Chem 276, 24525-24530.

13. Poenaru, S., Lamas, J. R., Folkers, G., Lopez de Castro, J. A., Seebach, D., and Rognan, D. (1999) J Med Chem 42, 2318-2331

14. Calbo, S., Guichard, G., Muller, S., Kourilsky, P., Briand, J. P., and Abastado, J. P. (2000) J Immunother 23, 125-130

15. Stemmer, C., Quesnel, A., Prevost-Blondel, A., Zimmermann, C., Muller, S., Briand, J. P., and Pircher, H. (1999) J Biol Chem 274, 5550-5556

16. Ostankovitch, M., Guichard, G., Connan, F., Muller, S., Chaboissier, A., Hoebeke, J., Choppin, J., Briand, J. P., and Guillet, J. G. (1998) J Immunol 161, 200-208

17. Meziere, C., Viguier, M., Dumortier, H., Lo-Man, R., Leclerc, C., Guillet, J. G., Briand, J. P., and Muller, S. (1997) J Immunol 159, 3230-3237


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

18. Jager, E., Chen, Y.-T., Drijfhout, J. W., Karbach, J., Ringhoffer, M., Jager, D., Arand, M., Wada, H., Noguchi, Y., Stockert, E., Old, L. J., and Knuth, A. (1998) J. Exp. Med.187, 265-270

19. Gnjatic, S., Atanackovic, D., Jager, E., Matsuo, M., Selvakumar, A., Altorki, N. K., Maki, R. G., Dupont, B., Ritter, G., Chen, Y. T., Knuth, A., Old, L. J., Zeng, G., Li, Y., El-Gamil, M., Sidney, J., Sette, A., Wang, R. F., Rosenberg, S. A., Robbins, P. F., Zarour, H. M., Maillere, B., Brusic, V., Coval, K., Williams, E., Pouvelle-Moratille, S., Castelli, F., Land, S., Bennouna, J., Logan, T., Kirkwood, J. M., Wang, X., Storkus, W. J., Touloukian, C. E., and Restifo, N. P. (2003) Proc Natl Acad Sci U S A100, 8862-8867

20. Gnjatic, S., Jager, E., Chen, W., Altorki, N. K., Matsuo, M., Lee, S. Y., Chen, Q., Nagata, Y., Atanackovic, D., Chen, Y. T., Ritter, G., Cebon, J., Knuth, A., and Old, L. J. (2002) Proc Natl Acad Sci U S A 99, 11813-11818

21. Valmori, D., Dutoit, V., Lienard, D., Rimoldi, D., Pittet, M. J., Champagne, P., Ellefsen, K., Sahin, U., Speiser, D., Lejeune, F., Cerottini, J. C., and Romero, P. (2000) Cancer Res 60, 4499-4506.

22. Romero, P., Dutoit, V., Rubio-Godoy, V., Lienard, D., Speiser, D., Guillaume, P., Servis, K., Rimoldi, D., Cerottini, J. C., and Valmori, D. (2001) Clin Cancer Res 7,766s-772s

23. Jager, E., Nagata, Y., Gnjatic, S., Wada, H., Stockert, E., Karbach, J., Dunbar, P. R., Lee, S. Y., Jungbluth, A., Jager, D., Arand, M., Ritter, G., Cerundolo, V., Dupont, B., Chen, Y. T., Old, L. J., and Knuth, A. (2000) Proc Natl Acad Sci U S A 97, 4760-4765.

24. Dutoit, V., Taub, R. N., Papadopoulos, K. P., Talbot, S., Keohan, M. L., Brehm, M., Gnjatic, S., Harris, P. E., Bisikirska, B., Guillaume, P., Cerottini, J. C., Hesdorffer, C. S., Old, L. J., and Valmori, D. (2002) J Clin Invest 110, 1813-1822

25. Chen, J.-L., Dunbar, P. R., Gileadi, U., Jager, E., Gnjatic, S., Nagata, Y., Stockert, E., Panicali, D. L., Chen, Y.-T., Knuth, A., Old, L. J., and Cerundolo, V. (2000) JImmunol 165, 948-955

26. Zeng, G., Li, Y., El-Gamil, M., Sidney, J., Sette, A., Wang, R. F., Rosenberg, S. A., and Robbins, P. F. (2002) Cancer Res 62, 3630-3635

27. Zarour, H. M., Maillere, B., Brusic, V., Coval, K., Williams, E., Pouvelle-Moratille, S., Castelli, F., Land, S., Bennouna, J., Logan, T., and Kirkwood, J. M. (2002) Cancer Res 62, 213-218

28. Jager, E., Gnjatic, S., Nagata, Y., Stockert, E., Jager, D., Karbach, J., Neumann, A., Rieckenberg, J., Chen, Y.-T., Ritter, G., Hoffman, E., Arand, M., Old, L. J., and Knuth, A. (2000) PNAS 97, 12198-12203

29. Valmori, D., Dutoit, V., Lienard, D., Rimoldi, D., Pittet, M. J., Champagne, P., Ellefsen, K., Sahin, U., Speiser, D., Lejeune, F., Cerottini, J.-C., and Romero, P. (2000) Cancer Res 60, 4499-4506


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

21

30. Garboczi, D. N., Madden, D. R., and Wiley, D. C. (1994) J Mol Biol 239, 581-587

31. Pflugrath, J. W. (1999) Acta Crystallogr D Biol Crystallogr 55, 1718-1725.

32. CCP4. (1994) Acta Crystallogr. D50, 750-763

33. Reid, S. W., McAdam, S., Smith, K. J., Klenerman, P., O'Callaghan, C. A., Harlos, K., Jakobsen, B. K., McMichael, A. J., Bell, J. I., Stuart, D. I., and Jones, E. Y. (1996) JExp Med 184, 2279-2286.

34. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr D Biol Crystallogr 54 ( Pt 5), 905-921

35. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard. (1991) Acta Crystallogr A 47( Pt 2), 110-119

36. Kjer-Nielsen, L., Clements, C. S., Brooks, A. G., Purcell, A. W., Fontes, M. R., McCluskey, J., and Rossjohn, J. (2002) J Immunol 169, 5153-5160

37. Chang, L., Kjer-Nielsen, L., Flynn, S., Brooks, A. G., Mannering, S. I., Honeyman, M. C., Harrison, L. C., McCluskey, J., and Purcell, A. W. (2003) Tissue Antigens 62,408-417

38. Chen, W., Yewdell, J. W., Levine, R. L., and Bennink, J. R. (1999) J Exp Med 189,1757-1764

39. Khan, A. R., Baker, B. M., Ghosh, P., Biddison, W. E., and Wiley, D. C. (2000) JImmunol 164, 6398-6405.

40. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C. (1987) Nature 329, 506-512

41. Saper, M. A., Bjorkman, P. J., and Wiley, D. C. (1991) J Mol Biol 219, 277-319

42. Madden, D. R., Garboczi, D. N., and Wiley, D. C. (1993) Cell 75, 693-708

43. Collins, E. J., Garboczi, D. N., and Wiley, D. C. (1994) Nature 371, 626-629

44. Bouvier, M., Guo, H. C., Smith, K. J., and Wiley, D. C. (1998) Proteins 33, 97-106

45. Hillig, R. C., Coulie, P. G., Stroobant, V., Saenger, W., Ziegler, A., and Hulsmeyer, M. (2001) J Mol Biol 310, 1167-1176

46. Sliz, P., Michielin, O., Cerottini, J. C., Luescher, I., Romero, P., Karplus, M., and Wiley, D. C. (2001) J Immunol 167, 3276-3284

47. Sylvester-Hvid, C., Kristensen, N., Blicher, T., Ferre, H., Lauemoller, S. L., Wolf, X. A., Lamberth, K., Nissen, M. H., Pedersen, L. O., and Buus, S. (2002) Tissue Antigens59, 251-258.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

22

48. Morgan, C. S., Holton, J. M., Olafson, B. D., Bjorkman, P. J., and Mayo, S. L. (1997) Protein Sci 6, 1771-1773

49. Rudolph, M. G., Speir, J. A., Brunmark, A., Mattsson, N., Jackson, M. R., Peterson, P. A., Teyton, L., and Wilson, I. A. (2001) Immunity 14, 231-242

50. Baratin, M., Kayibanda, M., Ziol, M., Romieu, R., Briand, J. P., Guiller, J. G., and Viguier, M. (2002) J Pept Sci 8, 327-334

51. Terasawa, H., Tsang, K. Y., Gulley, J., Arlen, P., and Schlom, J. (2002) Clin Cancer Res 8, 41-53

52. Blanchet, J. S., Valmori, D., Dufau, I., Ayyoub, M., Nguyen, C., Guillaume, P., Monsarrat, B., Cerottini, J. C., Romero, P., and Gairin, J. E. (2001) J Immunol 167,5852-5861

53. Micheletti, F., Bazzaro, M., Canella, A., Marastoni, M., Traniello, S., and Gavioli, R. (1999) Immunology 96, 411-415

54. van der Burg, S. H., Visseren, M. J., Brandt, R. M., Kast, W. M., and Melief, C. J. (1996) J Immunol 156, 3308-3314

55. Levitsky, V., Zhang, Q. J., Levitskaya, J., and Masucci, M. G. (1996) J Exp Med 183,915-926

56. Lipford, G. B., Bauer, S., Wagner, H., and Heeg, K. (1995) Vaccine 13, 313-320

57. Sette, A., Vitiello, A., Reherman, B., Fowler, P., Nayersina, R., Kast, W. M., Melief, C. J. M., Oseroff, C., Yuan, L., Ruppert, J., Sidney, J., Delguercio, M. F., Southwood, S., Kubo, R. T., Chesnut, R. W., Grey, H. M., and Chisari, F. V. (1994) J. Immunol.153, 5586-5592

58. Huarte, E., Sarobe, P., Lu, J., Casares, N., Lasarte, J. J., Dotor, J., Ruiz, M., Prieto, J., Celis, E., and Borras-Cuesta, F. (2002) Clin Cancer Res 8, 2336-2344

59. Apostolopoulos, V., Yu, M., Corper, A. L., Teyton, L., Pietersz, G. A., McKenzie, I. F., Wilson, I. A., and Plebanski, M. (2002) J Mol Biol 318, 1293-1305

60. Brooks, J. M., Colbert, R. A., Mear, J. P., Leese, A. M., and Rickinson, A. B. (1998) JImmunol 161, 5252-5259

61. Deng, Y., Yewdell, J. W., Eisenlohr, L. C., and Bennink, J. R. (1997) J Immunol 158,1507-1515

62. Lamas, J. R., Brooks, J. M., Galocha, B., Rickinson, A. B., and Lopez de Castro, J. A. (1998) Int Immunol 10, 259-266

63. Meadows, L., Wang, W., den Haan, J. M., Blokland, E., Reinhardus, C., Drijfhout, J. W., Shabanowitz, J., Pierce, R., Agulnik, A. I., Bishop, C. E., Hunt, D. F., Goulmy, E., and Engelhard, V. H. (1997) Immunity 6, 273-281


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

23

64. van der Most, R., Sette, A., Oseroff, C., Alexander, J., Murali-Krishna, K., Lau, L., Southwood, S., Sidney, J., Chesnut, R., Matloubian, M., and Ahmed, R. (1996) JImmunol 157, 5543-5554

65. Gold, J. S., Ferrone, C. R., Guevara-Patino, J. A., Hawkins, W. G., Dyall, R., Engelhorn, M. E., Wolchok, J. D., Lewis, J. J., and Houghton, A. N. (2003) J Immunol170, 5188-5194

66. Ercolini, A. M., Machiels, J. P., Chen, Y. C., Slansky, J. E., Giedlen, M., Reilly, R. T., and Jaffee, E. M. (2003) J Immunol 170, 4273-4280

67. Slansky, J. E., Rattis, F. M., Boyd, L. F., Fahmy, T., Jaffee, E. M., Schneck, J. P., Margulies, D. H., and Pardoll, D. M. (2000) Immunity 13, 529-538

68. Tangri, S., Ishioka, G. Y., Huang, X., Sidney, J., Southwood, S., Fikes, J., and Sette, A. (2001) J Exp Med 194, 833-846

69. Guichard, G., Calbo, S., Muller, S., Kourilsky, P., Briand, J. P., and Abastado, J. P. (1995) J Biol Chem 270, 26057-26059


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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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Wild type C9S C9A



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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


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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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.

10.1074/jbc.M314066200Access the most updated version of this article at doi:

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Functional and structural characteristics of NY-ESO-1 ... · vaccination with NY-ESO-1 determinants has been very promising, but along the way these studies have highlighted problems