Peptide-binding Motif Research Articles

Carbamazepine-Mediated Adverse Drug Reactions: CBZ-10,11-epoxide but Not Carbamazepine Induces the Alteration of Peptides Presented by HLA-B∗15:02.

Among patients treated with the anticonvulsive and psychotropic drug carbamazepine (CBZ), approximately 10% develop severe and life-threatening adverse drug reactions. These immunological conditions are resolved upon withdrawal of the medicament, suggesting that the drug does not manifest in the body in long term. The HLA allele B∗15:02 has been described to be a genomic biomarker for CBZ-mediated immune reactions. It is not well understood if the immune reactions are triggered by the original drug or by its metabolite carbamazepine-10,11-epoxide (EPX) and how the interaction between the drug and the distinct HLA molecule occurs. Genetically engineered human B-lymphoblastoid cells expressing soluble HLA-B∗15:02 molecules were treated with the drug or its metabolite. Functional pHLA complexes were purified; peptides were eluted and sequenced. Applying mass spectrometric analysis, CBZ and EPX were monitored by analyzing the heavy chain and peptide fractions separately for the presence of the drug. This method enabled the detection of the drug in a biological situation post-pHLA assembly. Both drugs were bound to the HLA-B∗15:02 heavy chain; however, solely EPX altered the peptide-binding motif of B∗15:02-restricted peptides. This observation could be explained through structural insight; EPX binds to the peptide-binding region and alters the biochemical features of the F pocket and thus the peptide motif. Understanding the nature of immunogenic interactions between CBZ and EPX with the HLA immune complex will guide towards effective and safe medications.

Influence of Genetic Polymorphism Towards Pulmonary Tuberculosis Susceptibility.

Tuberculosis (TB) is still remains the major threat for human health worldwide. Several case-control, candidate-gene, family studies and genome-wide association studies (GWAS) suggested the association of host genetic factors to TB susceptibility or resistance in various ethnic populations. Moreover, these factors modulate the host immune responses to tuberculosis. Studies have reported genetic markers to predict TB development in human leukocyte antigen (HLA) and non-HLA genes like killer immunoglobulin-like receptor (KIR), toll-like receptors (TLRs), cytokine/chemokines and their receptors, vitamin D receptor (VDR) and SLC11A1 etc. Highly polymorphic HLA loci may influence antigen presentation specificities by modifying peptide binding motifs. The recent meta-analysis studies revealed the association of several HLA alleles in particular class II HLA-DRB1 with TB susceptibility and valuable marker for disease development especially in Asian populations. Case-control studies have found the association of HLA-DR2 in some populations, but not in other populations, this could be due to an ethnic specific association of gene variants. Recently, GWAS conducted in case-control and family based studies in Russia, Chinese Han, Morocco, Uganda and Tanzania revealed the association of genes such as ASAP1, Alkylglycerol monooxygenase (AGMO), Forkhead BoxP1 (FOXP1), C-terminal domain phosphatase 1 (UBLCP1) and intergenic SNP rs932347C/T with TB. Whereas, SNP rs10956514A/G were not associated with TB in western Chinese Han and Tibetan population. In this review, we summarize the recent findings of genetic variants with susceptibility/resistance to TB.

Analysis of the affinity of influenza A virus protein epitopes for swine MHC I by a modified in vitro refolding method indicated cross-reactivity between swine and human MHC I specificities.

In vitro refolding assays can be used to investigate the affinity and stability of the binding of epitope peptides to major histocompatibility complex (MHC) class I molecules, which are key factors in the presentation of peptides to cytotoxic T lymphocytes (CTLs). The recognition of peptide epitopes by CTLs is crucial for protection against influenza A virus (IAV) infection. The peptide-binding motif of the swine SLA-3*hs0202 molecule has been previously reported and partly overlaps with the binding motif of the most abundant human MHC allele, HLA-A*0201. In this study, we screened all the protein sequences of the swine-origin epidemic IAV strain A/Beijing/01/2009 (H1N1), and a total of 73 9-mer epitope peptides were predicted to fit the consensus motif of the swine SLA-3*hs0202 or HLA-A*0201 molecule. Then, 14 peptides were selected, and their affinities to SLA-3*hs0202 were tested by a modified in vitro refolding assay. Our results show that ten epitopes could tolerate gel filtration, indicating that these epitopes formed stable or partly stable complexes with SLA-3*hs0202. Eight out of the ten epitopes have been previously reported as HLA-A2-restricted epitopes, which implied cross-reactivity between swine and human MHC I specificities. Furthermore, the modified mini-system refolding method could be applied for the screening of peptides because the refolding efficiency remained almost unchanged with the positive peptide (HA-KMN9) subjected to size-exclusion chromatography and Resource Q anion-exchange chromatography. The results presented here provide new insight into the development of epitope-based vaccines to control IAV and increase our understanding of swine molecular immunology.

Novel Structural Modification Based on Evans Blue Dye to Improve Pharmacokinetics of a Somastostatin-Receptor-Based Theranostic Agent.

The development of somastatin (SS) peptide analogues for the detection and treatment of neuroendocrine tumors has been successful with the recent FDA approval of 68Ga-DOTA-TATE and 177Lu-DOTA-TATE. The structure of these peptide constructs contains the peptide binding motif that binds to the receptor with high affinity, a chelator to complex the radioactive metal, and a linker between the peptide and chelator. However, these constructs suffer from rapid blood clearance, which limits their tumor uptake. In this study, this design has been further improved by incorporating a modification to control the in vivo pharmacokinetics. Adding a truncated Evans Blue (EB) dye molecule into the construct provides a prolonged half-life in blood as a result of its low micromolar affinity to albumin. We compared 177Lu-DOTA-TATE to the modified 177Lu Evans Blue compound (177Lu-DMEB-TATE), in vitro and in vivo in mice bearing A427-7 xenografts. The tumor uptake of 177Lu-DMEB-TATE was significantly greater than the uptake of 177Lu-DOTA-TATE in the biodistribution and SPECT-imaging studies. The therapeutic effect of the 177Lu-DMEB-TATE construct was superior to the that of the 177Lu-DOTA-TATE construct at the doses evaluated.

Major Histocompatibility Complex Class I (FLA-E*01801) Molecular Structure in Domestic Cats Demonstrates Species-Specific Characteristics in Presenting Viral Antigen Peptides.

Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag positions 40 to 48; RMANVSTGR [RMA9]). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular-dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and in vitro refolding of the mutant FLA-E*01801 complex demonstrated that Glu63 and Trp167 in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine.IMPORTANCE Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC-I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp167 blocks the left side of pocket A, causing P1D to conflict with Glu63 We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We identified 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC-I molecules present peptides.

Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.

Specificity Characterization of SLA Class I Molecules Binding to Swine-Origin Viral Cytotoxic T Lymphocyte Epitope Peptides in Vitro.

Swine leukocyte antigen (SLA) class I molecules play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. They mainly bind and present antigens of intracellular origin to circulating MHC I-restricted cytotoxic T lymphocytes (CTLs). Binding of an appropriate epitope to an SLA class I molecule is the single most selective event in antigen presentation and the first step in the killing of infected cells by CD8+ CTLs. Moreover, the antigen epitopes are strictly restricted to specific SLA molecules. In this study, we constructed SLA class I complexes in vitro comprising viral epitope peptides, the extracellular region of the SLA-1 molecules, and β2-microglobulin (β2m) using splicing overlap extension polymerase chain reaction (SOE-PCR). The protein complexes were induced and expressed in an Escherichia coli prokaryotic expression system and subsequently purified and refolded. Specific binding of seven SLA-1 proteins to one classical swine fever virus (CSFV) and four porcine reproductive and respiratory syndrome virus (PRRSV) epitope peptides was detected by enzyme-linked immunosorbent assay (ELISA)-based method. The SLA-1∗13:01, SLA-1∗11:10, and SLA-1∗11:01:02 proteins were able to bind specifically to different CTL epitopes of CSFV and PRRSV and the MHC restrictions of the five epitopes were identified. The fixed combination of Asn151Val152 residues was identified as the potentially key amino acid residues influencing the binding of viral several CTL epitope peptides to SLA-1∗13:01 and SLA-1∗04:01:01 proteins. The more flexible pocket E in the SLA-1∗13:01 protein might have fewer steric limitations and therefore be able to accommodate more residues of viral CTL epitope peptides, and may thus play a critical biochemical role in determining the peptide-binding motif of SLA-1∗13:01. Characterization of the binding specificity of peptides to SLA class I molecules provides an important basis for epitope studies of infectious diseases in swine, and for the rational development of novel porcine vaccines, as well as for detailed studies of CTL responses in pigs used as animal models.

A Specialist Macaque MHC Class I Molecule with HLA-B*27–like Peptide-Binding Characteristics

In different macaque species, the MHC A2*05 gene is present in abundance, and its gene products are characterized by low cell-surface expression and a highly conserved peptide-binding cleft. We have characterized the peptide-binding motif of Mamu-A2*05:01, and elucidated the binding capacity for virus-derived peptides. The macaque A2*05 allotype prefers the basic amino acid arginine at the second position of the peptide, and hydrophobic and polar amino acids at the C-terminal end. These preferences are shared with HLA-B*27 and Mamu-B*008, molecules shown to be involved in elite control in human HIV type 1 and macaque SIV infections, respectively. In contrast, however, Mamu-A2*05 preferentially binds 8-mer peptides. Retention in the endoplasmic reticulum seems to be the cause of the lower cell-surface expression. Subsequent peptide-binding studies have illustrated that Mamu-A2*05:01 is able to bind SIV-epitopes known to evoke a strong CD8+ T cell response in the context of the Mamu-B*008 allotype in SIV-infected rhesus macaques. Thus, the macaque A2*05 gene encodes a specialized MHC class I molecule, and is most likely transported to the cell surface only when suitable peptides become available.

The molecular basis for peptide repertoire selection in the human leukocyte antigen (HLA) C*06:02 molecule

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Structural Definition of Duck Major Histocompatibility Complex Class I Molecules That Might Explain Efficient Cytotoxic T Lymphocyte Immunity to Influenza A Virus.

ABSTRACTA single dominantly expressed allele of major histocompatibility complex class I (MHC I) may be responsible for the duck's high tolerance to highly pathogenic influenza A virus (HP-IAV) compared to the chicken's lower tolerance. In this study, the crystal structures of duck MHC I (Anpl-UAA*01) and duck β2-microglobulin (β2m) with two peptides from the H5N1 strains were determined. Two remarkable features were found to distinguish the Anpl-UAA*01 complex from other known MHC I structures. A disulfide bond formed by Cys95 and Cys112 and connecting the β5 and β6 sheets at the bottom of peptide binding groove (PBG) in Anpl-UAA*01 complex, which can enhance IAV peptide binding, was identified. Moreover, the interface area between duck MHC I and β2m was found to be larger than in other species. In addition, the two IAV peptides that display distinctive conformations in the PBG, B, and F pockets act as the primary anchor sites. Thirty-one IAV peptides were used to verify the peptide binding motif of Anpl-UAA*01, and the results confirmed that the peptide binding motif is similar to that of HLA-A*0201. Based on this motif, approximately 600 peptides from the IAV strains were partially verified as the candidate epitope peptides for Anpl-UAA*01, which is a far greater number than those for chicken BF2*2101 and BF2*0401 molecules. Extensive IAV peptide binding should allow for ducks with this Anpl-UAA*01 haplotype to resist IAV infection.IMPORTANCE Ducks are natural reservoirs of influenza A virus (IAV) and are more resistant to the IAV than chickens. Both ducks and chickens express only one dominant MHC I locus providing resistance to the virus. To investigate how MHC I provides IAV resistance, crystal structures of the dominantly expressed duck MHC class I (pAnpl-UAA*01) with two IAV peptides were determined. A disulfide bond was identified in the peptide binding groove that can facilitate Anpl-UAA*01 binding to IAV peptides. Anpl-UAA*01 has a much wider recognition spectrum of IAV epitope peptides than do chickens. The IAV peptides bound by Anpl-UAA*01 display distinctive conformations that can help induce an extensive cytotoxic T lymphocyte (CTL) response. In addition, the interface area between the duck MHC I and β2m is larger than in other species. These results indicate that HP-IAV resistance in ducks is due to extensive CTL responses induced by MHC I.

Synthesis and Optical Properties of Highly Stabilized Peptide-Coated Silver Nanoparticles

The interaction between peptide and gold nanoparticle surfaces has been increasingly of interest for bionanotechnology applications. To fully understand how to control such interactions, we have studied the optical properties of peptide-modified gold nanoparticles. However, the impacts of peptide binding motif upon the surface characteristics and physicochemical properties of nanoparticles remain poorly understood. Here, we have prepared sodium citrate-stabilized gold nanoparticles and coated with peptide IVD (ID3). These nanomaterials were characterized by UV-visible, transmission electron microscopy (TEM), and z-potential measurement. The results indicate that gold-peptide interface is generated using ID3 peptide and suggested that the reactivity of peptide is governed by the conformation of the bound peptide on the nanoparticle surface. The peptide-nanoparticle interactions could potentially be used to make specific functionality into the peptide capped nanomaterials.

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Peptidomic analysis of type 1 diabetes associated HLA-DQ molecules and the impact of HLA-DM on peptide repertoire editing.

HLA-DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII-bound peptides in human embryonic kidney 293T cells expressing HLA-DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM-resistant, DM-dependent, or DM-sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ-peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T-cell epitopes to DM editing.

Characterization of the Canine MHC Class I DLA-88*50101 Peptide Binding Motif as a Prerequisite for Canine T Cell Immunotherapy.

There are limitations in pre-clinical settings using mice as a basis for clinical development in humans. In cancer, similarities exist between humans and dogs; thus, the dog patient can be a link in the transition from laboratory research on mouse models to clinical trials in humans. Knowledge of the peptides presented on MHC molecules is fundamental for the development of highly specific T cell-based immunotherapies. This information is available for human MHC molecules but is absent for the canine MHC. In the present study, we characterized the binding motif of dog leukocyte antigen (DLA) class I allele DLA-88*50101, using human C1R and K562 transfected cells expressing the DLA-88*50101 heavy chain. MHC class I immunoaffinity-purification revealed 3720 DLA-88*50101 derived peptides, which enabled the determination of major anchor positions. The characterized binding motif of DLA-88*50101 was similar to HLA-A*02:01. Peptide binding analyses on HLA-A*02:01 and DLA-88*50101 via flow cytometry showed weak binding of DLA-88*50101 derived peptides to HLA-A*02:01, and vice versa. Our results present for the first time a detailed peptide binding motif of the canine MHC class I allelic product DLA-88*50101. These data support the goal of establishing dogs as a suitable animal model for the evaluation and development of T cell-based cancer immunotherapies, benefiting both dog and human patients.

Biochemical functionalization of peptide nanotubes with phage displayed peptides

The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

Structural and Biochemical Analyses of Swine Major Histocompatibility Complex Class I Complexes and Prediction of the Epitope Map of Important Influenza A Virus Strains.

The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. SLA-3*hs0202 binding with sβ2m and a KMNTQFTAV (hemagglutinin [HA]-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune responses. Notably, we found that different HA-KMN9 conformations are caused, not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG), but also by the skewing of α1 and α2 helixes forming the PBG. In addition, alanine scanning and circular-dichroism (CD) spectra confirmed that the B, D, and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC-I) structures, the fundamental motif for SLA-3*hs0202 was determined to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine. We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previous findings showing that variable peptide conformations are caused only by the flexibility of the side chains in the groove, the skewing of the α1 and α2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

BIITE: A Tool to Determine HLA Class II Epitopes from T Cell ELISpot Data.

Activation of CD4+ T cells requires the recognition of peptides that are presented by HLA class II molecules and can be assessed experimentally using the ELISpot assay. However, even given an individual’s HLA class II genotype, identifying which class II molecule is responsible for a positive ELISpot response to a given peptide is not trivial. The two main difficulties are the number of HLA class II molecules that can potentially be formed in a single individual (3–14) and the lack of clear peptide binding motifs for class II molecules. Here, we present a Bayesian framework to interpret ELISpot data (BIITE: Bayesian Immunogenicity Inference Tool for ELISpot); specifically BIITE identifies which HLA-II:peptide combination(s) are immunogenic based on cohort ELISpot data. We apply BIITE to two ELISpot datasets and explore the expected performance using simulations. We show this method can reach high accuracies, depending on the cohort size and the success rate of the ELISpot assay within the cohort.

Pooled screening for antiproliferative inhibitors of protein-protein interactions.

Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreas cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads and it provides a rich dataset covering phenotypes for inhibition of thousands of interactions.

Sequence diversity between class I MHC loci of African native and introduced Bos taurus cattle in Theileria parva endemic regions: in silico peptide binding prediction identifies distinct functional clusters.

There is strong evidence that the immunity induced by live vaccination for control of the protozoan parasite Theileria parva is mediated by class I MHC-restricted CD8(+) T cells directed against the schizont stage of the parasite that infects bovine lymphocytes. The functional competency of class I MHC genes is dependent on the presence of codons specifying certain critical amino acid residues that line the peptide binding groove. Compared with European Bos taurus in which class I MHC allelic polymorphisms have been examined extensively, published data on class I MHC transcripts in African taurines in T. parva endemic areas is very limited. We utilized the multiplexing capabilities of 454 pyrosequencing to make an initial assessment of class I MHC allelic diversity in a population of Ankole cattle. We also typed a population of exotic Holstein cattle from an African ranch for class I MHC and investigated the extent, if any, that their peptide-binding motifs overlapped with those of Ankole cattle. We report the identification of 18 novel allelic sequences in Ankole cattle and provide evidence of positive selection for sequence diversity, including in residues that predominantly interact with peptides. In silico functional analysis resulted in peptide binding specificities that were largely distinct between the two breeds. We also demonstrate that CD8(+) T cells derived from Ankole cattle that are seropositive for T. parva do not recognize vaccine candidate antigens originally identified in Holstein and Boran (Bos indicus) cattle breeds.

Abstract A075: Immunotherapy of malignant pleural mesothelioma using cMET specific T-cells

Abstract Background: Malignant pleural mesothelioma (MPM) is an incurable cancer predominantly induced by asbestos exposure. This aggressive tumour commonly presents at an advanced stage of disease. Although surgery, combination chemotherapy and radiotherapy may be used in disease management, median survival from diagnosis is less than 12 months. New therapeutic approaches are required to improve outcome in this disease. Chimeric antigen receptors (CAR) are fusion molecules that couple the HLA- independent binding of a selected cell surface target antigen to the delivery of a tailored T-cell activating signal. Using CAR technology, we aim to develop a novel immunotherapy for MPM that is both safe and effective. These molecules are delivered to patient T-cells using retroviral gene-transfer, thereby stably re-directing their specificity for antigen. The receptor tyrosine kinase MET is overexpressed in >80% of MPM patients making it an attractive candidate for CAR directed immunotherapy. Methods: Three second-generation cMet-targeted CARs (containing a fused CD28/CD3ζ endodomain) have been developed, distinguished by peptide-binding motifs. Functionality of MET-re targeted T-cells has been assessed by co-cultivation of genetically enhanced test and control T-cells with MPM cell lines that express MET at various levels. Results: Studies to date have shown that human T-cells engineered to express cMet CARs can destroy mesothelioma tumour cell monolayers, accompanied by T-cell proliferation and cytokine production. To further potentiate the activity of the cMet targeted CARs, a dual targeting approach to both cMet and the ErbB family is currently under evaluation. The second advantage of this approach is in diminishing the potential toxicity risks associate with “on target, off tumour” toxicity. An MPM xenograft model has been established utilising bioluminescence to further evaluate functionality and toxicity in vivo. Conclusions: These findings demonstrate proof of concept for the utility of CAR engineered T-cells to recognise and destroy cMet-expressing MPM tumour cells. We envisage that regional i.e. intrapleural delivery of human CAR positive T-cells could be used to maximise therapeutic index of this approach. Citation Format: Thivyan Thayaparan, Sjoukje J.C. van der Stegen, Ana C. Parente Pereira Puri, Roseanna Maria Petrovic, James Spicer, John Maher. Immunotherapy of malignant pleural mesothelioma using cMET specific T-cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A075.