High-throughput discovery of cancer-targeting TCRs.

The high-throughput discovery of T cell receptors (TCR) enables us to follow the longitudinal course of tumor disease, to monitor immunotherapy-related repertoire alterations, and exploit TCRs for therapeutic use. Here we discuss state of the art and future experimental and computational strategies to dissect the hypervariable world of TCRs and their corresponding antigens in the context of cancer immunotherapy.

Identification and isolation of antigen-specific cytotoxic T lymphocytes with an automated microraft sorting system.

The simultaneous measurement of T cell function with recovery of individual T cells would greatly facilitate characterizing antigen-specific responses both in vivo and in model systems. We have developed a microraft array methodology that automatically measures the ability of individual T cells to kill a population of target cells and viably sorts specific cells into a 96-well plate for expansion. A human T cell culture was generated against the influenza M1p antigen. Individual microrafts on a 70 × 70 array were loaded with on average 1 CD8+ cell from the culture and a population of M1p presenting target cells. Target cell killing, measured by fluorescence microscopy, was quantified in each microraft. The rates of target cell death among the individual CD8+ T cells varied greatly; however, individual T cells maintained their rates of cytotoxicity throughout the time course of the experiment enabling rapid identification of highly cytotoxic CD8+ T cells. Microrafts with highly active CD8+ T cells were individually transferred to wells of a 96-well plate, using a needle-release device coupled to the microscope. Three sorted T cells clonally expanded. All of these expressed high-avidity T cell receptors for M1p/HLA*02:01 tetramers, and 2 of the 3 receptors were sequenced. While this study investigated single T cell cytotoxicity rates against simple targets with subsequent cell sorting, future studies will involve measuring T cell mediated cytotoxicity in more complex cellular environments, enlarging the arrays to identify very rare antigen specific T cells, and measuring single cell CD4+ and CD8+ T cell proliferation.

Isolation and epitope mapping of staphylococcal enterotoxin B single-domain antibodies.

Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better.  Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper.

BACKGROUND: Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. STUDY DESIGN AND METHODS: Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Le(a) trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. RESULTS: Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. CONCLUSIONS: All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems.

Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002.

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ∼80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.

Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance.

We have developed a biomimetic sensor for the detection of human immunodeficiency virus type 1 (HIV-1) related protein (glycoprotein 41, gp41) based on epitope imprinting technique. gp41 is the transmembrane protein of HIV-1 and plays an important role in membrane fusion between viruses and infected cells. It is an important index for determining the extent of HIV-1 disease progression and the efficacy of therapeutic intervention. In this work, dopamine was used as the functional monomer and polymerized on the surface of quartz crystal microbalance (QCM) chip in the presence of template, a synthetic peptide with 35 amino acid residues, analogous to residues 579-613 of the gp41. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the QCM chip. QCM measurement showed that the resulting MIP film not only had a great affinity towards the template peptide, but also could bind the corresponding gp41 protein specifically. The dissociation constant (K(d)) of MIP for the template peptide was calculated to be 3.17 nM through Scatchard analysis, which was similar to those of monoclonal antibodies. Direct detection of the gp41 was achieved quantitatively using the resulting MIP-based biomimetic sensor. The detection limit of gp41 was 2 ng/mL, which was comparable to the reported ELISA method. In addition, the practical analytical performance of the sensor was examined by evaluating the detection of gp41 in human urine samples with satisfactory results.

Mass spectrometric and peptide chip epitope mapping of rheumatoid arthritis autoantigen RA33.

The protein termed RA33 was determined to be one major autoantigen in rheumatoid arthritis (RA) patients and antiRA33 auto-antibodies were found to appear shortly after onset of RA. They are often detectable before a final diagnosis can be made in the clinic. The aim of our study is to characterise the epitope of a monoclonal antiRA33 antibody on recombinant RA33 using mass spectrometric epitope mapping. Recombinant RA33 has been subjected to BrCN cleavage and fragments were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequent in-gel proteolytic digestion and mass spectrometric analysis determined the partial sequences in the protein bands. Western blotting of SDS-PAGE-separated protein fragments revealed immuno-positive, i.e. epitope-containing bands. BrCN-derived RA33 fragments were also separated by high- performance liquid chromatography (HPLC) and immuno-reactivity of peptides was measured by dot-blot analysis with the individual HPLC fractions after partial amino acid sequences were determined. The epitope region identified herewith was compared to data from peptide chip analysis with 15-meric synthetic peptides attached to a glass surface. Results from all three analyses consistently showed that the epitope of the monoclonal antiRA33 antibody is located in the aa79-84 region on recombinant RA33; the epitope sequence is MAARPHSIDGRVVEP. Sequence comparisons of the 15 best scoring peptides from the peptide chip analysis revealed that the epitope can be separated into two adjacent binding parts. The N-terminal binding parts comprise the amino acid residues "DGR", resembling the general physico-chemical properties "acidic/polar-small-basic". The C-terminal binding parts contain the amino acid residues "VVE", with the motif "hydrophobic-gap-acidic". The matching epitope region that emerged from our analysis on both the full-length protein and the 15-meric surface bound peptides suggests that peptide chips are indeed suitable tools for screening patterns of autoantibodies in patients suffering from autoimmune diseases.

Epitope mapping of human chromogranin A by peptide microarrays.

In this chapter we report on the characterization of linear antigenic sites of human chromogranin A (CgA), a useful tissue and serum marker for neuroendocrine tumours and a precursor of many biologically active peptides. The epitope mapping of CgA has been carried out by peptide microarrays on glass slides coated by a copolymer of N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS) and [3-(methacryloyl-oxy) propyl] trimethoxysilyl (MAPS). The microarray support provided sufficient accessibility of the ligand, with no need for a spacer, as the polymer chains prevent interaction of immobilized peptides with substrate. In addition, the polymeric surface constitutes an aqueous micro-environment in which, despite peptide random orientation, linear epitopes are freely exposed. The results reported are in accordance with those obtained in conventional ELISA assays using biotinylated and non-biotinylated peptides.

Peptide microarrays for serum antibody diagnostics.

Peptide microarrays are useful instruments for miniaturized high-throughput, high-content immunoassays. The substitution of linear epitopes of the protein antigen with short, synthetic peptides is virtually a straightforward approach to capture antigen-specific antibodies from serum samples; however, both the biologically active surface display of peptides and the establishment of a solidly performing peptide microarray immunoassay are often troublesome in detail. The following protocols aim to provide facilitated access to the production of a robust peptide microarray platform and an optimized analytical processing of peptide microarrays in serological diagnosis. The functional surface display is accomplished by site-specific immobilization of peptide probes in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde-coated substrate for microstructured surface immobilization of the probe complexes. The serological assay is based on the specific capture of primary antibodies in a sandwich complex between the surface-immobilized peptides and a fluorescently labelled secondary antibody. A proposal for the diagnostic evaluation of fluorescence data obtained with the peptide microarray is also made.

Evaluation of antigen-specific T-cell responses with a miniaturized and automated method.

The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-microl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-gamma) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10(4) PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.

Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

Comparison between the surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) method in a structural analysis of human endothelin-1.

In this study, an automated surface plasmon resonance (SPR)-based biosensor was compared with a quartz crystal microbalance (QCM) biosensor. The two biosensor systems were used for characterizing a site-directed monoclonal antibody (mAb), raised against the C-terminal heptapeptide ET-1(15-21) of the human endothelin (ET-1). The mAb was characterized by its capacity for binding to ET-1, ET-3, Big.ET-1(22-38), the C-terminal (ET-1(15-21), ET-1(16-21), ET-1(17-21)), and six derivates of ET-1(16-21), each containing a substitution with alanine (Ala) of a single aminoacid from position 16-21, respectively. The mAb reacted well with ET-1 and its fragments ET-1(15-21), ET-1(16-21), ET-1(17-21), but showed only a partial cross-reaction with ET-3, and did not bind human Big.ET-1(22-38). The Ala substitution on position 16,17, or 19 of ET-1(16-21) did not affect the antibody binding capacity of the hexapaptide ET-1(16-21). On the contrary, Ala substitution or Asp(18), Ile(20) and particularly Trp(21), inhibited its immunoreactivity. Thus the C-terminal represents an immunodominant epitope in ET-1 and is important for antibody binding. The SPR and QCM response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. With regard to the fundamental problem of comparing different measurement principles, we found a good correlation between results obtained using the BIA technology and the QCM.

Studies of Listeria monocytogenes-antibody binding using electro-orientation.

An electro-optical (EO) approach has been used for studies of Listeria monocytogenes-antibody binding. The EO analyzer, which has been developed at the State Research Center for Applied Microbiology, Obolensk, was used as a basic instrument for EO measurements. AC electro-kinetic effects depend on dielectric properties of bioparticles, their composition, morphology, the medium, and the frequency of applied electrical field. Electro-orientational spectra were used for discrimination of bacteria before and after selective binding with antibodies. The measurements were performed using a discrete set of frequencies of the orienting electric field (10, 100, 250, and 500 kHz). During biospecific interactions an antibody is bound to the microorganism causing a change in the dielectric properties of the microorganism-antibody complex and the EO signal reaches its maximum at 100-200 kHz. It has been shown that the biospecific interactions of L. monocytogenes cells with anti-Listeria antibody in the presence of Escherichia coli K-12, and Azospirillum brasilense Sp7 change the EO signals significantly. Thus, the determination of the presence of particular bacteria within a mixed sample may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing spectra of bacterium in the presence and in the absence of specific antibody.

Localization of T cell epitope regions of chicken ovomucoid recognized by mice.

We localized the T cell epitope regions of chicken ovomucoid (OVM), a potent egg allergen, with the overlapping pin-peptides covering the entire sequence of OVM and three strains of mice with different haplotypes. In C3H/He (H-2k) mice, the T cells recognized relatively broad regions on OVM; the dominant regions were 49-93 and 97-114 residues, and the subdominant regions were 7-21, 37-48, 94-96, 115-123 and 145-177 residues. In contrast, a more limited number of T cell epitope regions were localized in BALB/c (H-2d) and C57BL/6 (H-2b) mice. The T cells from BALB/c mice recognized 100-114 and 157-171 residues, and the T cells from C57BL/6 mice recognized only 157-180 residues. These results were confirmed by using peptides separately synthesized and purified on the putative epitope regions. The roles of the carbohydrate moieties and cysteine residues involved in the disulfide bridges of OVM were also examined, and we found that they were not important in recognition by the T cell/antigen presenting cell.

Combinatorial peptide library methods for immunobiology research.

The field of combinatorial peptide chemistry has emerged as a powerful tool in the study of many biological systems. This review focuses on combinatorial peptide library methodology, which includes biological library methods, spatially addressable parallel library methods, library methods requiring deconvolution, the "one-bead one-compound" library method, and affinity chromatography selection method. These peptide libraries have successfully been employed to study a vast array of cell surface receptors, as well as have been useful in identifying protein kinase substrates and inhibitors. In recent immunobiological applications, peptide libraries have proven monumental in the definition of MHC anchor residues, in lymphocyte epitope mapping, and in the development of peptide vaccines. Peptides identified from such libraries, when presented in a chemical microarray format, may prove useful in immunodiagnostics. Combinatorial peptide libraries offer a high-throughput approach to study limitless biological targets. Peptides discovered from such studies may be therapeutically and diagnostically useful agents.

Epitope-mapping of transglutaminase with parallel label-free optical detection.

The gastrointestinal disorder coeliac disease (CD) is induced by the ingestion of wheat gluten and is characterized by damage of the typical structure of the intestinal mucosa. The enzyme tissue transglutaminase (tTGase) was identified as the major target of disease-specific antibodies in-patients. We performed an epitope fine-mapping with a series of pentadecapeptides synthesized using parallel multiple peptide synthesis. For the detection of biomolecular interactions a label-free parallel method, reflectometric interference spectroscopy (RIfS), was used. This is the first optical label-free method adapted to a high throughput screening (HTS) format and the experimental results demonstrate its applicability as a biological screening device. A high titer of anti-tTGase antibodies is found in the serum of coeliac patients. We have taken the first step towards a fast non-surgical test for the detection of these antibodies. In order to identify and characterize a continuous epitope with high affinity against the anti-tTGase antibody a screening of 21 pentadecapeptides has been accomplished with the parallel RIfS system. A single channel RIfS-system with high resolution was used to determine binding constants of identified peptides with high affinity.

The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports--principles and applications.

Presented first in 1990 at the 21st European Peptide Symposium in Barcelona, Spain [Frank, R., Güler, S., Krause, S., Lindenmaier, W., 1991. Facile and rapid 'spot synthesis' of large numbers of peptides on membrane sheets. In: Giralt, E., Andreu, D. (Eds.) Peptides 1990, Proc. 21st Eur. Peptide Symp. ESCOM, Leiden, p. 151.], the SPOT-synthesis method opened up countless opportunities to synthesise and subsequently screen large numbers of synthetic peptides as well as other organic compounds arrayed on a planar cellulose support [Tetrahedron 48 (1992) 9217]. Already in 1991, a commercial kit for manual SPOT-synthesis became available through Cambridge Research Biochemicals (CRB, UK), and in 1993, a semi-automated SPOT-synthesiser, the ASP222, was launched by ABIMED Analysen-Technik, Germany. Both made the technique available to many research laboratories, even those not experienced in or equipped for chemistry. Although SPOT-synthesis is not as impressively miniaturised as, e.g. the Affymax photolithographic technique [Science 251 (1991) 767], it fulfils similar demands with the advantage of a reliable and easy experimental procedure, inexpensive equipment needs and a highly flexible array and library formatting. The method permits rapid and highly parallel synthesis of huge numbers of peptides and peptide mixtures (pools) including a large variety of unnatural building blocks, as well as a growing range of other organic compounds. Further advantages are related to the easy adaptability to a wide range of assay and screening methods such as binding, enzymatic and cellular assays, which allow in situ screening of chemical libraries due to the special properties of the membrane supports. Therefore, peptide arrays prepared by the SPOT-technique became quite popular tools for studying numerous aspects of molecular recognition, particularly in the field of molecular immunology.

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein.

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.