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J. Biol. Chem., Vol. 279, Issue 22, 23438-23446, May 28, 2004
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From the
Protein Crystallography Unit and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia, ¶T cell Laboratory, Ludwig Institute for Cancer Research, Austin and Repatriation Medical Centre, Heidelberg, Victoria 3084, Australia, and the **Department of Microbiology and Immunology and ImmunoID, University of Melbourne, Parkville, Victoria 3010, Australia
Received for publication, December 23, 2003 , and in revised form, March 2, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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. More often, TAAs are self-proteins overexpressed in tumors or self-proteins that contain mutations that may or may not be discernible 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 toward 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 toward 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 (residues 155-163, QLSLLMWIT; residues 157-165, SLLMWITQC; residues 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 mainly recognize the NY-ESO-(157-165) determinant (21). The immunogenicity of these peptides was first evaluated in a trial vaccination of cancer patients in which a mixture of the peptides was administered intradermally to patients bearing NY-ESO-1+ tumors (28). A vigorous TCD8 response to NY-ESO-(157-165) was observed, whereas reactivity against NY-ESO-(157-165) 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-ESO-(157-165) 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-ESO-(157-165) where the C-terminal Cys residue has been replaced with more conventional anchor residues, namely Leu9 and Val9 analogs, have been generated (25). Whereas these analogs bind more efficiently to HLA A2 and are recognized by CTL raised against the natural NY-ESO-(157-165) peptide, they do not induce effective anti-tumor CTL. Indeed, the presence of the Cys at the C terminus seems critical for generating CTL that recognize endogenously processed NY-ESO determinants on tumor cells. The presence of this amino acid causes problems with formulation due to oxidative damage and dimerization, both of which reduce the efficacy of the peptide Ag as an immunogen (25). In this study, we have investigated the structure of NY-ESO-(157-165) complexed to HLA A*0201 and compared it with the C9A and C9S structures, which are more easily formulated and are potential vaccine candidates (see Table I). 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-ESO-(157-165). In our studies, we have been careful to pretreat all of the peptides including the Cys-containing peptides with a reductant to prevent dimerization or cysteinylation of the peptides, which 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 used structure guided design to test an analog that should satisfy the Cys requirement of anti-tumor CTL by substituting the Cys9 for a nonnatural isosteric analog of this residue 2-aminoisobutyric acid (Abu).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Expression, Purification, Crystallization, and Structure Determination—Truncated HLA A*0201 class I heavy chain, encompassing residues 1-274, was expressed as inclusion bodies (30) using the BL21 (RIL) strain of Escherichia coli. At an A600 of 0.6, cultures were induced with 1 mM of isopropyl-1-thio--D-galactopyranoside for 12 h, bacteria were lysed in 50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 1% sodium deoxycholate, 100 mM NaCl, and 10 mM dithiothreitol. Inclusion bodies were isolated by centrifugation after washing with 50 mM Tris-HCl, 0.5% Triton X-100, 100 mM NaCl, 1 mM NaEDTA, 1 mM dithiothreitol, pH 8.0, and washing in 50 mM Tris-HCl, 1 mM NaEDTA, 1 mM dithiothreitol, pH 8.0, and then solubilized in 50 mM Tris, 8 M urea, 10 mM NaEDTA, pH 8.0, with the protease inhibitors 1 µg/ml pepstatin A and 200 µM phenylmethylsulfonyl fluoride. Recombinant protein (30 mg of A2 heavy chain and 10 mg of 2-microglobulin) was refolded with 6 mg of peptide reconstituted in 3 M guanidine HCl, 10 mM sodium acetate, and 10 mM NaEDTA, pH 4.2, in a refolding buffer composed of 0.1 M Tris, 2 mM EDTA, 400 mM L-arginine-HCl, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, pH 8.0, at 4 °C for 72 h. Following refolding, protein was dialyzed overnight against Milli Q using a 6,000-8,000-kDa molecular mass cut-off dialysis membrane (Spectrum). Protein was concentrated by ion exchange on a DE52 column (Whatman, Maidstone, Kent, UK) and subsequently purified by size exclusion on a Superdex 75pg gel filtration column (Amersham Biosciences) and a final ion exchange on a MonoQ HR 10/10 column (Amersham Biosciences). Quantitative analysis was based on comparisons with bovine serum albumin protein standards using SDS-PAGE. Protein was concentrated to 10 mg/ml for use in crystallization trials.
Crystallization—Large cubic crystals (0.3 x 0.3 x 0.3 mm) were obtained using the hanging drop vapor diffusion technique at room temperature. The crystals were grown within 3-5 days by mixing equal volumes of 10 mg/ml HLA A2-NY-ESO-1 peptide (and analogues thereof) with the reservoir buffer (2.0 M ammonium sulfate, 0.1 M sodium citrate, pH 6.5). The crystals belong to space group P213 with unit cell dimensions a = b = c 
117.90 Å, 
= = 
= 90°. The crystals were flash-frozen prior to data collection using crystals that had been soaked in 15% glycerol. One 2.2-Å and two 2.5-Å data sets were collected for the NY-ESO-1 series and scaled using the HKL suite (31). For a summary of statistics, see Table I.
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 (Protein Data Bank code 1DUY
[PDB]
) (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 Rfactor 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 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 3.5
, and had a B-factor less than 60 Å2. See Table I for a 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) was inserted into pET30 (Novagen) vector and verified by DNA sequencing. Inclusion body protein of the heavy chain and 2-microglobulin were prepared as described (30, 36, 37). In vitro assembly of HLA A2-peptide complexes in microassembly reactions was initiated by the sequential addition of recombinant 2-microglobulin (2 µM) and HLA A2 heavy chain (3 µM)to peptide (30 µM) in a buffer containing 100 mM Tris, pH 8.0, 0.4 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride in a final volume of 1 ml. The assembly reaction mixture was allowed to proceed at 4 °C for 48 h, and aggregated material was removed by centrifugation. Quantitation of assembled HLA class I complexes was performed by capture enzyme-linked immunosorbent assay; briefly, 96-well plates were coated with affinity-purified pan-class I-specific monoclonal antibody W6/32 at 10 µg/ml, washed three times with phosphate-buffered saline containing 0.05% Tween 20 (PBST), and blocked with PBST containing 1% bovine serum albumin. Properly assembled and correctly conformed HLA-peptide complexes were captured and detected by incubation with horseradish peroxidase-conjugated rabbit anti-human 2-microglobulin polyclonal antibodies (DakoCytomation A/S, Glostrup, Denmark) and the chromogen o-phenylenediamine (Sigma).
Thermostability Measurements of Recombinant Class I Complexes Using Circular Dichroism—CD spectra were measured on a Jasco 810 spectropolarimeter using a thermostatically controlled cuvette at temperatures between 20 and 90 °C. Far-UV spectra from 195 to 250 nm were collected with a 5-s/point signal averaging and were the accumulation of 10 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 inflection point, which represents the Tm for each protein. All complexes were measured at 20 µg/ml in a solution of 10 mM Tris, 150 mM NaCl, pH 8.0.
T Cell Lines and Interferon- Assay—The NY-ESO-1-specific CTL lines with specificity against NY-ESO-1-(157-165) were derived from delayed type hypersensitivity biopsy after HLA A2+ patients bearing NY-ESO+ tumors received NY-ESO-1 peptide 157-165 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 patients 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 tris-(2-carboxyethyl)-phosphine hydrochloride (Pierce), which reduces oxidation, dimerization, 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-deficient T2 cells were pulsed with graded concentrations of the peptides at room temperature for 45 min and then washed. T cells were then added along with brefeldin A at a final concentration of 10 µg/ml. The cells were incubated for a further 4 h, harvested, and stained with anti-CD8-Cychrome conjugate in 50 µl of phosphate-buffered saline at 4 °C for 30 min, washed, and fixed with 1% paraformaldehyde. The cells were permeabilized with 0.2% saponin and intracellular interferon- that had accumulated in the presence of brefeldin A was detected using an anti-interferon--fluorescein isothiocyanate conjugate. 100,000 events were acquired on a FACScalibur flow cytometer, and the data were analyzed with Flowjo software (TreeStar, San Carlos, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2-microglobulin, and nine residues of the bound peptide, one sulfate ion, and a variable number of water molecules.
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The N-terminal P1-Ser is strongly tethered within the cleft, with the main chain forming hydrogen bonds with the side chains of Tyr7, Tyr159, and Tyr171, whereas the side chain stacks against Trp167, and the P1-Ser O group forming a hydrogen bond with Glu63. Glu63 also forms a hydrogen bond with the main chain of P2-Leu, a hydrophobic anchor residue that correspondingly sits in the hydrophobic B pocket, comprising Tyr7, Phe9, Met45, Val67, and Tyr99 of the A2 heavy chain. Tyr99 also interacts with the P3-Leu side chain, a residue that also sits in a hydrophobic pocket. An abrupt alteration in the main chain conformation at P3-Leu (
= -65, 
= 154), P4-Met ( = -73, = -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 Arg97, although its guanadinium group is orientated away from this pocket, forming a salt bridge with Asp77, a residue located in the F-pocket (Fig. 3). In comparison with some other HLA A2 structures, the positioning of Arg97 is varied such that in a previously determined A2 complex (Protein Data Bank code 1DUY [PDB] ), Arg97 does not form a salt bridge with Asp77. Instead, Arg97 points "upward" toward the bulged section of the bound peptide. Arg97 is sandwiched between Tyr99 and Tyr116, with Tyr116 being orientated toward the D pocket. In our NY-ESO-(157-165) complex, Tyr116 is orientated toward the F-pocket. Thus, the positioning of Arg97 also impacts significantly on the positioning of Tyr116, a key F pocket residue.
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and C group forms van der Waals interactions with Trp147, whereas the sulfur moiety of the P9-Cys is neither in the correct geometry nor within suitable hydrogen-bonding distance to make hydrogen bond contacts with F pocket residues. Instead, the sulfur moiety exclusively forms van der Waals contacts with Thr143, Leu81, and Asp77.
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-ESO-(157-165)-HLA A2 complex. Comparison of the wild type with the C9A-HLA A2 complex yielded a root mean square deviation of 0.10 Å for the 383 C atoms. Comparison of the wild type with the C9S-HLA A2 complex yielded a root mean square deviation of 0.15 Å for the 383 C atoms. Variations 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 C of P9-Ser and P9-Cys. Additional alterations occur to accommodate the more polar Ser functionality, with the P9-Ser O making a direct hydrogen bond to Asp77 resulting in small movement of the hydroxyl group relative to the thiol group of P9-Cys. As discussed below, these subtle changes in F pocket binding lead to substantial changes in complex stability, suggesting that 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 der Waals interactions, we substituted the cysteine for Abu, a nonnatural 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 I). This analog was synthesized 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-ESO-(157-165) 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 enzyme-linked immunosorbent assay (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 0.5-10 µM, 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 CD. All complexes gave similar spectra at 20 °C; however, the midpoint thermal denaturation revealed compelling differences in the stability of these complexes (Fig. 5). C9V was 4.5 °C more stable than the wild type, whereas 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In this study, we were able to compare the relative immunogenicity of each analogue with 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 tris-(2-carboxyethyl)-phosphine hydrochloride 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 recognized more efficiently by the T cell lines, and as a general rule the following reactivity pattern was observed: C9Abu > C9A, C9V > wild type > 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 that 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 that 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 that 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, 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 et al. (63) that a peptide originating from SMCY was only recognized 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 a 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 lymphocytic choriomeningitis virus 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). Whereas this frequently imparts improved MHC binding, it does not always equate to improved immunity toward the naturally processed peptide in vivo. For example, our data clearly show that substitution for more appropriate P9 anchor residues for HLA A2 such as valine or leucine, while 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 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. (68) demonstrated the potential for heteroclitic epitopes in inducing high avidity cross-reactive anti-tumor CTL against tolerant or weakly immunogenic TAA based on conservative or semiconservative natural amino acid substitutions. As such, this report is one of few studies to successfully incorporate nonnatural amino acids into T cell epitopes (9-11, 29) and highlights the path ahead for rational vaccine design.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council, the Roche Organ Transplantation Research Foundation, and the Juvenile Diabetes Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
These two authors contributed equally to this work.
|| Supported by Wellcome Trust Senior Research Fellowships in Biomedical Science in Australia.
To whom correspondence and reprint requests may be addressed. Tel.: 613-9905-3736; Fax: 613-9905-4699; E-mail: jamie.rossjohn{at}med.monash.edu.au. ¶¶ A C.R. Roper Fellow of the Faculty of Medicine, Dentistry, and Health Science at the University of Melbourne. To whom correspondence and reprint requests may be addressed. Tel.: 613-8344-9911; Fax: 613-9347-1540; E-mail: apurcell{at}unimelb.edu.au.
1 The abbreviations used are: MHC, major histocompatibility complex; Ag, antigen; TCD8, CD8+ T lymphocytes; CTL, cytotoxic T lymphocyte(s); Fmoc, N-(9-fluorenyl)methoxycarbonyl; TAA, tumor-associated antigen(s); Abu, 2-aminoisobutyric acid.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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