Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.

Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.

Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.

Biochemical diversity in a phage display library of random decapeptides.

Detailed knowledge and understanding of the structure and function of biologically important macromolecules is frequently insufficient to permit rational, de novo design of recognition molecules and therapeutics. Traditional drug discovery has, thus, focused on screening and identifying native molecules as drugs or as templates for genetic engineering or organic synthesis. The number and novelty of lead compounds for drug discovery might be expanded significantly, however, by the ability to express and screen large libraries of peptide structures with phage display technologies [Scott and Smith, Science 249 (1990) 386-390]. The significance of such libraries as sources of novel biological ligands will depend in part on the depth and degree of biochemical diversity they comprise. We have prepared a phage display library of greater than 2 x 10(6) individual decapeptides produced as N-terminal fusions to the pIII surface protein of fd filamentous phage. The decapeptides were expressed from a degenerate DNA insert sequence that was chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons. Fifty-two clones were picked randomly and without prior selection from the population and the sequences of their peptide inserts were determined. Our results confirm and document the broad representation at the primary amino acid sequence level that is expected in a library expressed from random DNA inserts. More significantly, biochemical characterization shows these insert sequences correspond to structures comprising a wide range and combination of isoelectric, hydropathic, and biochemical properties necessary in drug discovery to access a significant percentage of the repertoire of possible peptide structures by affinity or activity screening.

Antigen-binding repertoire and Ig H chain gene usage among B cell hybridomas from normal and autoimmune mice.

LPS-stimulated B cells were used to generate a panel of mAb that were a random sample of the preimmune repertoire of C57BL/6 and highly autoimmune, viable motheaten mice. These mAb were tested for reactivity to a number of "self" and foreign Ag. Binding that could be detected only at nM mAb concentrations or less was considered significant. We found that a surprisingly high number of the mAb bound one or more of the Ag tested, and many mAb bound more than a single Ag. Ag-induced mAb were likewise tested and found to have greatly reduced cross-reactivities. We found no significant differences, either in frequency of Ag binding or degree of cross-reactivity, between normal and autoimmune mice. Furthermore, the frequency with which a given Ag was bound by our panel of mAb was found to be proportional to the size of the Ag. The frequency with which individual VH gene families were expressed by our panel was consistent with a stochastic usage of VH genes in the preimmune repertoire. We interpret these data as showing that the preimmune repertoire is highly cross-reactive and that the activation of autoreactive clones in autoimmune animals is due to a defect in cellular regulation rather than a difference in repertoire.

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus.

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.

A disconnection alarm for the Bennett BA-4 ventilator.

A simple warning device is described to attach to a Bennett BA-4 Ventilator. It has proved reliable in practice when the ventilator is operated on pressure limit.