A rapid method for quantitative prediction of high affinity CTL epitopes: QSAR studies on peptides having affinity with the class I MHC molecular HLA-A*0201

Summary  It would be useful to develop a method to rapidly identify peptide epitopes for vaccine development. In this paper, empirical three-dimensional quantitative structure-affinity relationship (3D-QSAR) methods were used to study the relationship between the three dimensional structural parameters (the isotropic surface area, ISA, and the electronic charge index, ECI) of the HLA-A^*0201 binding peptide and the HLA-A^*0201/peptide binding affinities. A set of 102 peptides having affinity with the class I MHC HLA-A^*0201 molecule was used as training set. A test set of 40 peptides was used to determine the predictive value of the models. The 3D-QSAR models gave aq ²=0.5724 and highr _pred ² =0.6955. According to the standard regression coefficients, it is known that the hydrophobic interactions (in these studies, the ISA is highly correlative with the hydrophobic property) play a dominant role in peptide-MHC molecule binding, and also which amino acid residue with what property is needed at specific position of the peptide. The approach we have taken is highly complementary to the many excellent methods described in references and appears highly predictive. It is a rapid and convenient method for detecting high affinity peptide epitopes.     

Toward the quantitative prediction of T-cell epitopes: QSAR studies on peptides having affinity with the class I MHC molecular HLA-A*0201.

It would be useful for vaccine development to develop a method of rapidly identifying peptide epitopes. In this paper, the empirical three-dimensional quantitative structure-affinity relationship (3D-QSAR) methods were used to study the relationship between the three dimensional structural parameters (the isotropic surface area, ISA, and the electronic charge index, ECI) of the HLA-A*0201 binding peptide and the HLA-A*0201/peptide binding affinities. A set of 102 peptides having affinity with the class I MHC HLA-A*0201 molecule was used as training set. A test set of 40 peptides was used to determine the predictive value of the models. The 3D-QSAR models yielded a q2 = 0.5724 and a high rpred2 = 0.6955. The standard regression coefficients indicated that the hydrophobic interactions played an important role in peptide-MHC molecule binding and predicted the specific amino acid residue essential at a certain position of the peptide. The approach tested in the current paper is highly complementary to many of the methods described in references and possesses good predictability. It is a rapid and convenient method to detect high affinity peptide epitopes.     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A^∗0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them—p248-256, p140-149 and p144-152—were found to bind to HLA-A^*0201 molecules with a high affinity and effectively induced a recall response of CD8⁺ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587⁺ tumors. Notably, these peptide-specific CD8⁺ T cells exhibited potent cytotoxic activity over HCA587⁺ tumor cells. Taken together, the present study has identified three new HLA-A^*0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

Two distinct HLA-A * 0101-specific submotifs illustrate alternative peptide binding modes

 Previous studies have defined two different peptide binding motifs specific for HLA-A ^* 0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A ^* 0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A ^* 0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE₃ submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A ^* 0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A ^* 0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A ^* 0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A ^* 0101-restricted peptide epitopes. Received: 16 July 1996 / Revised: 18 September 1996     

Different immunodominance of HIV-1-specific CTL epitopes among three subtypes of HLA-A*26 associated with slow progression to AIDS

It is speculated that HLA-A^∗26-restricted HIV-1-specific CTLs can control HIV-1, since HLA-A^∗26 is associated with a slow progression to AIDS. In three major HLA-A^∗26 subtypes, HLA-A^∗2601-restricted, and HLA-A^∗2603-restricted HIV-1 epitopes have been identified, but HLA-A^∗2602-restricted ones have not. We here identified HLA-A^∗2602-restricted HIV-1 epitopes by using reverse immunogenetics and compared the immunodominance of the epitopes among the three subtypes. Out of 110 HIV-1 peptides carrying HLA-A^∗26 anchor residues, only the Gag169-177 peptide, which had been previously identified as an HLA-A^∗2601- and HLA-A^∗2603-restricted immunodominant epitope, induced Gag169-177-specific CD8⁺ T cells from only two of six HLA-A^∗2602⁺ HIV-1-infected individuals. No difference in affinity of this epitope peptide was found among these three HLA-A^∗26 subtypes, indicating that Gag169-177 was effectively presented by HLA-A^∗2602 but recognized as a subdominant epitope in HIV-1-infected HLA-A^∗2602⁺ individuals. These findings indicate different immunodominance of Gag169-177 epitope among 3 HLA-A^∗26 subtypes.     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with N^ε-(5-carboxyfluorescein)-l -lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (K_a) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.     

Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995     

Quantitative approaches to computational vaccinology

This article reviews the newly released JenPep database and two new powerful techniques for T-cell epitope prediction: (i) the additive method; and (ii) a 3D-Quantitative Structure Activity Relationships (3D-QSAR) method, based on Comparative Molecular Similarity Indices Analysis (CoMSIA). The JenPep database is a family of relational databases supporting the growing need of immunoinformaticians for quantitative data on peptide binding to major histocompatibility complexes and to the Transporters associated with Antigen Processing (TAP). It also contains an annotated list of T-cell epitopes. The database is available free via the Internet (http://www.jenner.ac.uk/JenPep). The additive prediction method is based on the assumption that the binding affinity of a peptide depends on the contributions from each amino acid as well as on the interactions between the adjacent and every second side-chain. In the 3D-QSAR approach, the influence of five physicochemical properties (steric bulk, electrostatic potential, local hydrophobicity, hydrogen-bond donor and hydrogen-bond acceptor abilities) on the affinity of peptides binding to MHC molecules were considered. Both methods were exemplified through their application to the well-studied problem of peptides binding to the human class I MHC molecule HLA-A*0201.     

Coupling to the surface of liposomes alters the immunogenicity of hepatitis C virus-derived peptides and confers sterile immunity

We have previously demonstrated that antigens chemically coupled to the surface of liposomes consisting of unsaturated fatty acids were cross-presented by antigen presenting cells to cytotoxic T lymphocytes (CTLs). Liposomal form of immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus exhibited highly efficient antiviral CTL responses in immunized mice. In this study, we coupled 15 highly conserved immunodominant CTL epitope peptides derived from hepatitis C virus (HCV) to the surface of liposomes. We also emulsified the peptides in incomplete Freund’s adjuvant, and compared the immune responses of the two methods of presenting the peptides by cytotoxicity induction and interferon-gamma (IFN-γ) production by CD8⁺ T cells of the immunized mice. We noticed significant variations of the immunogenicity of each peptide between the two antigen delivery systems. In addition, the immunogenicity profiles of the peptides were also different from those observed in the mice infected with recombinant adenoviruses expressing HCV proteins as previously reported. Induction of anti-viral immunity by liposomal peptides was tested by the challenge experiments using recombinant vaccinia viruses expressing corresponding HCV epitopes. One D^b-restricted and three HLA-A^*0201-restricted HCV CTL epitope peptides on the surface of liposomes were found to confer complete protection to immunized mice with establishment of long-term memory. Interestingly, their protective efficacy seemed to correlate with the induction of IFN-γ producing cells rather than the cytotoxicity induction suggesting that the immunized mice were protected through non-cytolytic mechanisms. Thus, these liposomal peptides might be useful as HCV vaccines not only for prevention but also for therapeutic use.     

Molecular definition of an elusive third HLA-A9 molecule: HLA-A9.3

The HLA-A9 family has been characterized as possessing two well defined specificities; HLA-A23 and A24. Serological studies have suggested the presence of a third member of this family HLA-A9.3, however there is doubt surrounding the existence of this specificity. HLA-A23, A24, and the putative A9.3 proteins were analyzed biochemically by immunoprecipitation and isoelectric focusing. Both HLA-A24 and A9.3 have identical isoelectric points whereas A23 is different. We have sequenced cDNA encoding HLA-A23, A24, and A9.3. From the observed protein sequences, we found A9.3 to differ from A24 by two amino acid substitutions located in the ₂ helix of the class I molecule. These substitutions are expected to significantly change the shape of the peptide binding cleft.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M64740 (HLA-A ^*2402); M64741 (HLA-A ^*2403); M64742 (HLA-A ^*2301). Address correspondence and offprint requests to: P. Parham.     

Additive method for the prediction of protein-peptide binding affinity. Application to the MHC class I molecule HLA-A*0201

A method has been developed for prediction of binding affinities between proteins and peptides. We exemplify the method through its application to binding predictions of peptides with affinity to major histocompatibility complex class I molecule HLA-A*0201. The method is named "additive" because it is based on the assumption that the binding affinity of a peptide could be presented as a sum of the contributions of the amino acids at each position and the interactions between them. The amino acid contributions and the contributions of the interactions between adjacent side chains and every second side chain were derived using a partial least squares (PLS) statistical methodology using a training set of 420 experimental IC50 values. The predictive power of the method was assessed using rigorous cross-validation and using an independent test set of 89 peptides. The mean value of the residuals between the experimental and predicted pIC50 values was 0.508 for this test set. The additive method was implemented in a program for rapid T-cell epitope search. It is universal and can be applied to any peptide-protein interaction where binding data is known.     

Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1^*0501, β1^*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides. Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed.     

Structure-activity relationship of T-cell receptors based on alanine scanning

T-cell receptors (TCR) recognize complexes between human leukocyte antigens (HLA) and peptides derived from intracellular proteins. Their therapeutic use for antigen targeting, however, has been hindered by the very low binding affinity of TCRs, typically in the 1- to 100-μM range. Therefore, to construct mutant TCRs with high binding affinity, we need to understand the relationship between the structure and activity of these molecules. Here, we attempted to identify the amino acids of the TCR that are important for binding to the peptide/HLA complex. We used a TCR that recognizes complexes between HLA-A^∗0201 and the peptide from tyrosinase, antigen overexpressed in melanoma. We changed 16 amino acids in the third complementarity-determining region within the TCR to alanine and examined the effect on binding affinity. Five alanine substitutions decreased the binding affinity to below 10% compared with that of wild-type TCR. In contrast, one alanine substitution caused a faster on-rate and slower off-rate, and increased the binding affinity to three times that of the wild-type TCR. Our results provide fundamental information for constructing mutant TCRs with high binding affinity.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.