Antibody activity in ankylosing spondylitis sera to two sites on HLA B27.1 at the MHC groove region (within sequence 65-85), and to a Klebsiella pneumoniae nitrogenase reductase peptide (within sequence 181-199).

74 overlapping peptides of varying lengths from Klebsiella pneumoniae nitrogenase reductase (residues 181-199) and from the HLA B27.1 molecule (residues 65-85) were synthesized and tested by ELISA against sera from HLA B27+ ankylosing spondylitis (AS) patients, and sera from HLA B27+ and HLA B27- healthy first-degree relatives. Antibody activity in AS sera to Klebsiella peptides of four to eight amino acids was maximal with the peptide NSRQTDR. Activity to HLA B27 peptides was maximal with the peptide KAKAQTDR (named epitope I). These peptides overlap with, but are proximal to the NH2 terminus from QTDRED, which is homologous in HLA B27.1 and K. pneumoniae nitrogenase reductase. A second weaker reactive site was noted in the HLA B27.1 peptides, proximal to the COOH terminus from the homologous sequence, namely peptide REDLRTLL (named epitope II). Little activity was seen against peptides that included the entire homologous sequence. Sera from 50 AS patients showed higher total Ig activity against peptides KAKAQTDR (p less than 0.001) and NSRQTDR (p less than 0.02) than did sera from 22 B27+ and 22 B27- healthy controls. These data indicate that AS patient sera contain antibodies that bind to K. pneumoniae nitrogenase peptides and HLA B27.1 peptides, and that there are at least two epitopes on HLA B27.1 in the alpha 1 domain, at the MHC groove region, that are autoantigenic in AS patients. Epitope I may be a site for crossreactivity between HLA B27 and Klebsiella.

Recognition of multiple peptide cores by a single T cell receptor.

We present evidence that a single T cell clone can recognize at least five different overlapping peptides, each with its distinct core structure, in the context of the same major histocompatibility complex (MHC) molecule. Distinct core residues are crucial for triggering the T cell receptor (TCR) in each case. These results suggest that the TCR (a) has multiple sets of contact residues for alternative peptide-MHC ligands, the binding to any one of which can trigger the cell; and/or (b) is able to attach to the peptide-MHC complex in more than one orientation. In this sense, the TCR is a multisubsite structure capable of being stimulated by a variety of peptide ligands associated with the same MHC molecules.

T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.

Chemistry of antibody binding to a protein.

The chemistry of antibody recognition was studied by mapping the antigenicity of the protein myohemerythrin with peptide homologs of the protein sequence. The results suggest that the entire protein surface is antigenic, but the probability of there being antibodies to a given site is influenced by local stereochemistry. Although accessible to an antibody binding domain, the least reactive positions cluster in the most tightly packed and least mobile regions and are closely associated with narrow, concave grooves in the molecular surface containing bound water molecules. The most frequently recognized sites form three-dimensional superassemblies characterized by high local mobility, convex surface shape, and often by negative electrostatic potential.

Mechanisms of antibody binding to a protein.

The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

A priori delineation of a peptide which mimics a discontinuous antigenic determinant.

A technique was developed for identifying peptides with high affinity for a given antibody. By testing a monoclonal antibody directed against a discontinuous antigenic determinant on foot-and-mouth disease virus, peptides mimicking the determinant were identified even though the tertiary structure of the proteins comprising the virus capsid is unknown. The allowable variations in spacing and stereochemistry of the peptides shown to mimic this epitope suggest protein folding in which amino acid residues from three regions, distant from one another in the primary sequence, are brought into close proximity at this epitope. The technique has potential for identification of peptides which will bind with high affinity to receptors other than antibody molecules.

Similar binding properties of peptide ligands for a human immunoglobulin and its light chain dimer.

The urinary light chain dimer and serum monoclonal IgG1 protein from a patient (Mcg) with multiple myeloma and amyloidosis were systematically tested for their binding activities to peptides presented on solid supports. The system was validated using a series of enkephalins, beta-casomorphins and DNP-lysine derivatives which were known to complex with the dimer. Sets of peptide ligands binding to the proteins were constructed by incremental additions of amino acid residues to minimal binding units [Geysen et al., J. Immun. Meth. 102, 259-274 (1987)]. Both the amino acid sequences and the combinations of optical isomers were optimized at each stage of the syntheses. Binding could be demonstrated for ligands ranging in size from a tethered single amino acid to pentapeptides. At the dipeptide levels, the dimer and the IgG1 protein showed different preferences (Hp versus qf, where lower case letters designate D-amino acid residues). However, in a tetrapeptide ligand (qfHp) for the dimer, both of these initial preferences had converged. With few exceptions, the IgG1 molecule showed binding activity for the ligands developed for the dimer. Two sets of selected peptides, one based on Hp and the other on mW, were synthesized for diffusion into crystals of the dimer. X-ray analyses showed that these peptides bound exclusively in the main binding cavity between the "variable" domains of the dimer. As predicted from the ELISA results with tethered ligands, the relative occupancies in the crystals followed the order of tetrapeptide greater than tripeptide much greater than dipeptide. The crystallographic studies confirmed that peptides with very different sequences can bind in the same cavity.

The antibody response to myoglobin--I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants.

Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.

Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

Simultaneous multiple synthesis of peptide-carrier conjugates.

Recently, the multipin approach for simultaneous multiple peptide synthesis was applied to the analysis of T cell determinants by using a novel cleavage method (Maeji et al., 1990). A diketopiperazine forming linker allowed cleavage of peptides into aqueous buffer which, without further purification, could be used immediately in cell culture assays. Another potential application of the technique is the simultaneous cleavage and coupling of peptides to immunogenic carriers. Without further purification the resulting conjugates can be used for the production of antipeptide antisera. The choice of carrier and conjugation chemistry is not restricted as peptide/pin cleavage occurs in aqueous solution over a range of pH and ionic strength. The method was assessed using the 2,4-dinitrophenyl group as a model hapten, diphtheria toxoid as the carrier, and N-(epsilon-maleimidocaproyloxy)succinimide as the cross-linking reagent. The resulting DNP-DT conjugate was used to prepare high titered specific anti-DNP antisera in mice.

Multi-pin peptide synthesis strategy for T cell determinant analysis.

Techniques to synthesize many peptides simultaneously exist, however their individual cleavage and subsequent purification constitutes a bottleneck to total throughput. Biological screening of peptides is generally carried out at physiological pH in aqueous solutions. However, peptides, unless individually purified are usually contaminated by residual compounds used in their preparation such as trifluoroacetic acid, organic solvents, scavengers etc. In testing with cellular systems, such as T cell determinant analysis, such contaminations must be rigorously excluded. We have extended the pin synthesis technique of synthesizing and screening large number of peptides (Geysen et al., 1984) to the analysis of T cell determinants. Peptides can be synthesized on polyethylene pins, the side chain protective groups removed and the peptides washed free of contaminants. A linker system stable under these conditions can then be triggered to cleave the peptides from the pins in an aqueous solution at neutral pH. This strategy enables the rapid mapping of T cell determinants. It is also applicable to other systems where large numbers of solution phase peptides are required, for example, in the study of hormone analogues.

Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands.

The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide.

Strategies for epitope analysis using peptide synthesis.

A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens.

Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides.

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum.

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.

Ratio encoding combinatorial libraries with stable isotopes and their utility in pharmaceutical research.

Combinatorial libraries are an important tool for lead discovery in the pharmaceutical industry. Advances in high throughput screening coupled with combinatorial chemistry can significantly reduce the time to find lead compounds. A major difficulty in developing large combinatorial libraries is the ability to identify active compounds. This paper describes a rapid and sensitive encoding/decoding methodology that utilizes stable isotopes and mass spectrometry. The ability of mass spectrometry to precisely determine the intensity of isotopic abundances provides a unique encoding strategy employing synthetically generated ratios of stable isotopes in a compound as the code. The application of ratio encoding is demonstrated using peptoid and imidazole chemistries. Supporting data demonstrate that the incorporation of one or more stable isotopes using unique-predetermined ratios can encode chemical libraries. In addition, the presence of a unique isotopic pattern in a ligand can facilitate the pharmacokinetic analysis. Isotope incorporation into a compound and subsequently into its metabolites reliably distinguishes products from other molecules in the mass spectrum. This is illustrated by metabolic analyses of peptoid and imidazole compounds.

Characterization of a Plasmodium falciparum epitope recognized by a monoclonal antibody with broad isolate and species specificity.

Monoclonal antibody (MAb) 7H8 raised against Plasmodium yoelii reacted with a series of proteins from P. falciparum that range in molecular weight from 46 to 194 kDa. By immunofluorescence assay, this MAb reacted with all isolates of P. falciparum tested. MAb 7H8 was used to screen a genomic expression library of asexual blood stage antigens of P. falciparum, Malayan Camp K+ and 7 independent clones were identified. These 7 clones were sequenced and the epitope recognized by MAb 7H8 in the recombinant protein of one of these clones was mapped. This epitope contained Lys Tyr Pro as core amino acids. However, similar sequences were not found in the other clones, indicating that this MAb binds to a structural epitope formed by different amino acids. The variable composition of the epitope may account for the number of P. falciparum malarial proteins recognized by MAb 7H8.