Most pockets in the human leukocyte antigen-group DR (HLA-DR) groove are shaped by clusters of polymorphic residues and, thus, have distinct chemical and size characteristics in different HLA-DR alleles. Each HLA-DR pocket can be characterized by "pocket profiles," a quantitative representation of the interaction of all natural amino acid residues with a given pocket. In this report we demonstrate that pocket profiles are nearly independent of the remaining HLA-DR cleft. A small database of profiles was sufficient to generate a large number of HLA-DR matrices, representing the majority of human HLA-DR peptide-binding specificity. These virtual matrices were incorporated in software (TEPITOPE) capable of predicting promiscuous HLA class II ligands. This software, in combination with DNA microarray technology, has provided a new tool for the generation of comprehensive databases of candidate promiscuous T-cell epitopes in human disease tissues. First, DNA microarrays are used to reveal genes that are specifically expressed or upregulated in disease tissues. Second, the prediction software enables the scanning of these genes for promiscuous HLA-DR binding sites. In an example, we demonstrate that starting from nearly 20,000 genes, a database of candidate colon cancer-specific and promiscuous T-cell epitopes could be fully populated within a matter of days. Our approach has implications for the development of epitope-based vaccines.

SummaryWe have investigated whether sequence 67 to 74 shared by B chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRBI*0401, DRBI*0404, and the closely related, RA nonassociated DRBI*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRBI*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR ~ chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

SummaryWe describe here a new method for predicting class II major histocompatibility complex-binding peptides, based on the preferences observed in a systematic series of peptide binding experiments where each position in a "minimal" peptide was replaced individually by every amino acid. The DRBI*0401 peptide binding preferences were determined and incorporated into a computer program that looks through sequences for potential epitopes and assigns each a score. These scores correlate well with previously determined T cell epitopes of foreign antigens and endogenous peptides from self proteins. Our findings hold implications for the design of subunit vaccines and in the identification of autoantigenic peptide regions within self proteins.M HC class II molecules are highly polymorphic proteins that bind peptides derived from processing of antigens and present them to T cells (1). Methods to predict regions in protein sequences capable of binding to human MHC class II molecules would be very valuable for many immunological applications. A quantitative prediction of MHC binding sites, however, requires the understanding of the rules governing peptide--MHC class II interaction. The use of M13 peptide display libraries to identify aUele-specific motifs (2-4), together with structural analyses of HLA-DRl-peptide complexes (5, 6) provided the basis to predict MHC class II-peptide interaction: (a) the identification of precisely spaced anchor positions in large HLA-DR-selected peptide pools and the x-ray crystallographic studies suggested an overall similar, sequence-independent peptide conformation (3, 5); (b) the fact that the anchor at position 1 (pl) 1 was found obligatory for high affinity binding allowed us to define frames in HLA-DR bound peptides (4); (c) the position of a particular side chain, with respect to the pl-frame,'determines whether a given amino acid behaves as anchor residue, inhibitory residue, or residue neutral for binding (4). This information led us to develop a new method for predicting class II MHC-binding peptides. The method is based on the hypothesis that peptide binding to HLA-DR correlates with the net result of all side chain effects in any given peptide, and that most side chain effects depend on their relative position to the pl anchor, rather than on the remaining peptide se- quence. Pl-anchored designer peptide libraries were thus used for scanning the relative peptide positions 2-9 for the effects of each amino acid side chain on binding to the human class II molecule DRBI*0401. When values, individually obtained by side chain scanning, were assigned to the amino acid residues of the selected protein region, the sum of these values correlated with the MHC-binding affinity of the respective peptide. The processing of the side chain scanning data into a software allowed a rapid and nearly perfect prediction of DRBI*0401 binding regions in any protein sequence. Materials and MethodsPreparation of HLA-DRBI*0401 Molecules. DRBI*0401 molecules were isolated from human lymphoblastoid cell...

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized α-MSH peptide analogues, 4,7,4,7, In-DOTAGlyGlu-CycMSH by SPECT/CT images at 2 h post-injection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h post-injection. There was low radioactivity (<0.8 %ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively-charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h post-injection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using 111 In-labeled lactam bridge-cyclized α-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.