Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Functional and Structural Characteristics of NY-ESO-1-related HLA A2-restricted Epitopes and the Design of a Novel Immunogenic Analogue

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m314066200

ISSN

1083-351X

Autores

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, Anthony W. Purcell,

Tópico(s)

T-cell and B-cell Immunology

Resumo

NY-ESO-1, a commonly expressed tumor antigen of the cancer-testis family, is expressed by a wide range of tumors but not found in normal adult somatic tissue, making it an ideal cancer vaccine candidate. Peptides derived from NY-ESO-1 have shown preclinical and clinical trial promise; however, biochemical features of these peptides have complicated their formulation and led to heterogeneous immune responses. We have taken a rational approach to engineer an 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 reduced complex stability, resulting in poor cytotoxic T lymphocyte recognition. Conversely, substitution with alanine maintained complex stability and cytotoxic T lymphocyte recognition. Based on the structures of the three HLA A2 complexes, we incorporated 2-aminoisobutyric acid, an isostereomer of cysteine, into the epitope. This analogue is impervious to oxidative damage, cysteinylation, and dimerization of the peptide epitope upon formulation that is characteristic of the wild type peptide. Therefore, this approach has yielded a potential therapeutic molecule that satiates the hydrophobic F pocket of HLA A2 and exhibited superior immunogenicity relative to the wild type peptide. NY-ESO-1, a commonly expressed tumor antigen of the cancer-testis family, is expressed by a wide range of tumors but not found in normal adult somatic tissue, making it an ideal cancer vaccine candidate. Peptides derived from NY-ESO-1 have shown preclinical and clinical trial promise; however, biochemical features of these peptides have complicated their formulation and led to heterogeneous immune responses. We have taken a rational approach to engineer an 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 reduced complex stability, resulting in poor cytotoxic T lymphocyte recognition. Conversely, substitution with alanine maintained complex stability and cytotoxic T lymphocyte recognition. Based on the structures of the three HLA A2 complexes, we incorporated 2-aminoisobutyric acid, an isostereomer of cysteine, into the epitope. This analogue is impervious to oxidative damage, cysteinylation, and dimerization of the peptide epitope upon formulation that is characteristic of the wild type peptide. Therefore, this approach has yielded a potential therapeutic molecule that satiates the hydrophobic F pocket of HLA A2 and exhibited superior immunogenicity relative to the wild type peptide. Class I major histocompatibility complex (MHC) 1The 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.1The 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. 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 (1Boon T. Vanderbruggen P. J. Exp. Med. 1996; 183: 725-729Crossref PubMed Scopus (774) Google Scholar, 2Platsoucas 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. Fazilleau N. Anticancer Res. 2003; 23: 1969-1996PubMed Google Scholar, 3Jager D. Jager E. Knuth A. J. Clin. Pathol. 2001; 54: 669-674Crossref PubMed Scopus (152) Google Scholar, 4Engelhard V.H. Bullock T.N. Colella T.A. Sheasley S.L. Mullins D.W. Immunol. Rev. 2002; 188: 136-146Crossref PubMed Scopus (107) Google Scholar, 5Kourilsky P. Fazilleau N. Int. Rev. Immunol. 2001; 20: 575-591Crossref PubMed Scopus (2) Google Scholar). 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 (6Sette A. Fikes J. Curr. Opin. Immunol. 2003; 15: 461-470Crossref PubMed Scopus (257) Google Scholar). 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 nonpeptidic structures (7Purcell A.W. Zeng W. Mifsud N.A. Ely L.K. Macdonald W.A. Jackson D.C. J. Pept. Sci. 2003; 9: 255-281Crossref PubMed Scopus (49) Google Scholar, 8Guichard G. Connan F. Graff R. Ostankovitch M. Muller S. Guillet J.G. Choppin J. Briand J.P. J. Med. Chem. 1996; 39: 2030-2039Crossref PubMed Scopus (45) Google Scholar, 9Guichard G. Zerbib A. Le Gal F.A. Hoebeke J. Connan F. Choppin J. Briand J.P. Guillet J.G. J. Med. Chem. 2000; 43: 3803-3808Crossref PubMed Scopus (48) Google Scholar, 10Krebs S. Folkers G. Rognan D. J. Pept. Sci. 1998; 4: 378-388Crossref PubMed Scopus (11) Google Scholar, 11Krebs S. Lamas J.R. Poenaru S. Folkers G. de Castro J.A. Seebach D. Rognan D. J. Biol. Chem. 1998; 273: 19072-19079Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Several studies have incorporated nonnatural amino acids in peptidic structures to improve compound stability and maintain T cell cross-reactivity. For example, some studies have used nonnatural amino acids with modified side chains that approximate the natural amino acid (10Krebs S. Folkers G. Rognan D. J. Pept. Sci. 1998; 4: 378-388Crossref PubMed Scopus (11) Google Scholar, 11Krebs S. Lamas J.R. Poenaru S. Folkers G. de Castro J.A. Seebach D. Rognan D. J. Biol. Chem. 1998; 273: 19072-19079Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) or by modifying peptide bonds by introducing β-amino acids (12Reinelt S. Marti M. Dedier S. Reitinger T. Folkers G. de Castro J.A. Rognan D. J. Biol. Chem. 2001; 276: 24525-24530Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 13Poenaru S. Lamas J.R. Folkers G. Lopez de Castro J.A. Seebach D. Rognan D. J. Med. Chem. 1999; 42: 2318-2331Crossref PubMed Scopus (39) Google Scholar), reducing peptide bonds from the natural amine bonds to aminomethylene (14Calbo S. Guichard G. Muller S. Kourilsky P. Briand J.P. Abastado J.P. J. Immunother. 2000; 23: 125-130Crossref PubMed Scopus (14) Google Scholar, 15Stemmer C. Quesnel A. Prevost-Blondel A. Zimmermann C. Muller S. Briand J.P. Pircher H. J. Biol. Chem. 1999; 274: 5550-5556Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) or generation of partially modified retro-inverso pseudopeptides (8Guichard G. Connan F. Graff R. Ostankovitch M. Muller S. Guillet J.G. Choppin J. Briand J.P. J. Med. Chem. 1996; 39: 2030-2039Crossref PubMed Scopus (45) Google Scholar, 16Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.P. Guillet J.G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar, 17Meziere C. Viguier M. Dumortier H. Lo-Man R. Leclerc C. Guillet J.G. Briand J.P. Muller S. J. Immunol. 1997; 159: 3230-3237PubMed Google Scholar). 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 (1Boon T. Vanderbruggen P. J. Exp. Med. 1996; 183: 725-729Crossref PubMed Scopus (774) Google Scholar). 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 (18Jager E. Chen Y.-T. Drijfhout J.W. Karbach J. Ringhoffer M. Jager D. Arand M. Wada H. Noguchi Y. Stockert E. Old L.J. Knuth A. J. Exp. Med. 1998; 187: 265-270Crossref PubMed Scopus (635) Google Scholar). 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 (19Gnjatic 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. Restifo N.P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8862-8867Crossref PubMed Scopus (184) Google Scholar, 20Gnjatic 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. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11813-11818Crossref PubMed Scopus (81) Google Scholar, 21Valmori D. Dutoit V. Lienard D. Rimoldi D. Pittet M.J. Champagne P. Ellefsen K. Sahin U. Speiser D. Lejeune F. Cerottini J.C. Romero P. Cancer Res. 2000; 60: 4499-4506PubMed Google Scholar, 22Romero P. Dutoit V. Rubio-Godoy V. Lienard D. Speiser D. Guillaume P. Servis K. Rimoldi D. Cerottini J.C. Valmori D. Clin. Cancer Res. 2001; 7: 766s-772sPubMed Google Scholar, 23Jager 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. Knuth A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4760-4765Crossref PubMed Scopus (355) Google Scholar, 24Dutoit 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. Valmori D. J. Clin. Invest. 2002; 110: 1813-1822Crossref PubMed Scopus (86) Google Scholar, 25Chen 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. Cerundolo V. J. Immunol. 2000; 165: 948-955Crossref PubMed Scopus (142) Google Scholar, 26Zeng G. Li Y. El-Gamil M. Sidney J. Sette A. Wang R.F. Rosenberg S.A. Robbins P.F. Cancer Res. 2002; 62: 3630-3635PubMed Google Scholar, 27Zarour H.M. Maillere B. Brusic V. Coval K. Williams E. Pouvelle-Moratille S. Castelli F. Land S. Bennouna J. Logan T. Kirkwood J.M. Cancer Res. 2002; 62: 213-218PubMed Google Scholar). 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 (21Valmori D. Dutoit V. Lienard D. Rimoldi D. Pittet M.J. Champagne P. Ellefsen K. Sahin U. Speiser D. Lejeune F. Cerottini J.C. Romero P. Cancer Res. 2000; 60: 4499-4506PubMed Google Scholar, 23Jager 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. Knuth A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4760-4765Crossref PubMed Scopus (355) Google Scholar, 28Jager 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. Knuth A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12198-12203Crossref PubMed Scopus (398) Google Scholar). 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 (18Jager E. Chen Y.-T. Drijfhout J.W. Karbach J. Ringhoffer M. Jager D. Arand M. Wada H. Noguchi Y. Stockert E. Old L.J. Knuth A. J. Exp. Med. 1998; 187: 265-270Crossref PubMed Scopus (635) Google Scholar). Despite poor binding to HLA A2, tumor-reactive TCD8 clones mainly recognize the NY-ESO-(157-165) determinant (21Valmori D. Dutoit V. Lienard D. Rimoldi D. Pittet M.J. Champagne P. Ellefsen K. Sahin U. Speiser D. Lejeune F. Cerottini J.C. Romero P. Cancer Res. 2000; 60: 4499-4506PubMed Google Scholar). 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 (28Jager 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. Knuth A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12198-12203Crossref PubMed Scopus (398) Google Scholar). 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 (24Dutoit 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. Valmori D. J. Clin. Invest. 2002; 110: 1813-1822Crossref PubMed Scopus (86) Google Scholar). 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 (20Gnjatic 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. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11813-11818Crossref PubMed Scopus (81) Google Scholar). 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 (25Chen 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. Cerundolo V. J. Immunol. 2000; 165: 948-955Crossref PubMed Scopus (142) Google Scholar). 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 (25Chen 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. Cerundolo V. J. Immunol. 2000; 165: 948-955Crossref PubMed Scopus (142) Google Scholar). 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 (24Dutoit 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. Valmori D. J. Clin. Invest. 2002; 110: 1813-1822Crossref PubMed Scopus (86) Google Scholar) 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).Table IPeptides used in this study Peptides—All peptides were synthesized using standard Fmoc synthesis and synthesized by Auspep Pty. Ltd. (North Melbourne, Victoria, Australia). All peptides were purified to >85% purity by preparative reverse phase high pressure liquid chromatography, and purity was determined by liquid chromatography-mass spectrometry using an Agilent 1100 LC-MSD SL ion trap instrument and a Stable Bond RP C18 column (100 × 0.5-mm inner diameter column) (see Table I). Peptides were dissolved in Me2SO to a final concentration of 10-100 mg/ml. Expression, Purification, Crystallization, and Structure Determination—Truncated HLA A*0201 class I heavy chain, encompassing residues 1-274, was expressed as inclusion bodies (30Garboczi D.N. Madden D.R. Wiley D.C. J. Mol. Biol. 1994; 239: 581-587Crossref PubMed Scopus (31) Google Scholar) using the BL21 (RIL) strain of Escherichia coli. At an A600 of 0.6, cultures were induced with 1 mm of isopropyl-1-thio-β-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 β2-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 × 0.3 × 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, α = β = γ = 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 (31Pflugrath J.W. Acta Crystallogr. D. Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1410) Google Scholar). 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 (32Collaborative Computing Project 4Acta Crystallogr. Sect. D. 1994; 50: 750-763Google Scholar). The previously solved monomeric HLA A2 structure (Protein Data Bank code 1DUY) (33Reid 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. Jones E.Y. J. Exp. Med. 1996; 184: 2279-2286Crossref PubMed Scopus (116) Google Scholar), 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) (34Brunger 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. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar), interspersed with rounds of model building using the program O (35Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). 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 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 β2-microglobulin were prepared as described (30Garboczi D.N. Madden D.R. Wiley D.C. J. Mol. Biol. 1994; 239: 581-587Crossref PubMed Scopus (31) Google Scholar, 36Kjer-Nielsen L. Clements C.S. Brooks A.G. Purcell A.W. Fontes M.R. McCluskey J. Rossjohn J. J. Immunol. 2002; 169: 5153-5160Crossref PubMed Scopus (67) Google Scholar, 37Chang L. Kjer-Nielsen L. Flynn S. Brooks A.G. Mannering S.I. Honeyman M.C. Harrison L.C. McCluskey J. Purcell A.W. Tissue Antigens. 2003; 62: 408-417Crossref PubMed Scopus (17) Google Scholar). In vitro assembly of HLA A2-peptide complexes in microassembly reactions was initiated by the sequential addition of recombinant β2-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 β2-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; θ218 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-γ 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 (38Chen W. Yewdell J.W. Levine R.L. Bennink J.R. J. Exp. Med. 1999; 189: 1757-1764Crossref PubMed Scopus (97) Google Scholar). 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-γ that had accumulated in the presence of brefeldin A was detected using an anti-interferon-γ-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). 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 2.5 Å or better. The structures were determined via molecular replacement, using a previously determined HLA A2 structure as the search probe (Protein Data Bank number 1DUY (39Khan A.R. Baker B.M. Ghosh P. Biddison W.E. Wiley D.C. J. Immunol. 2000; 164: 6398-6405Crossref PubMed Scopus (145) Google Scholar)). The structure of HLA A*0201 complexed to the wild type NY-ESO-(157-165) peptide has been refined to 2.2 Å to an Rfactor and Rfree of 22.8 and 26.7% respectively; the structure of the C9A analogue has been refined to 2.3 Å to an Rfactor and Rfree of 23.6 and 27.3%, respectively; the structure of the C9S analogue has been refined to 2.5 Å to an Rfactor and Rfree of 23.0 and 27.9%, respectively (See Table II 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.Table IIData collection and refinement statistics The values in parentheses are for the highest r

Referência(s)