Majority of Peptides Binding HLA-A*0201 With High Affinity Crossreact With Other A2-Supertype Molecules John Sidney, Scott Southwood, Dean L. Mann, Marcelo A. Fernandez-Vina, Mark J. Newman, and Alessandro Sette ABSTRACT: The A*0201, A *0202, A*0203, A*0206, and A*6802 binding capacity of single amino acid sub- stitution analogs of known A2-supertype binding pep- tides and of large nonredundant peptide libraries was measured. The results were utilized to rigorously define the peptide binding specificities of these A2-supertype molecules. Although each molecule was noted to have unique preferences, large overlaps in specificity were found. The presence of L, I, V, M, A, T, and Q residues in position 2, and L, I, V, M, A, and T residues at the C-terminus of peptide ligands were tolerated by all mol- ecules. Likewise, whereas examination of secondary influ- ences on peptide binding revealed allele specific prefer- ences, shared features could also be identified. These shared features were utilized to define an A2-supermotif and were noted to correlate with crossreactivity. Over 70% of the peptides that bound A *0201 with high affinity were found to bind at least two other A2-super- type molecules. Because the A2-supertype molecules studied herein cover the variants most common in differ- ent major ethnicities, these findings have important im- plications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune re- sponses. Human Immunology 62, 1200 –1216 (2001). © American Society for Histocompatibility and Immu- nogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: MHC; antigens; peptides; epitopes; anti- gen binding; immunochemistry; CTL INTRODUCTION Human leukocyte antigen (HLA) class I molecules are cell surface glycoproteins encoded by the major histo- compatibility complex (MHC) cluster of genes whose main biologic function is to bind antigenic peptides (epitopes) and present them to T cells [1]. HLA mole- cules are known to be extremely polymorphic, and, in fact, the 1996 WHO HLA Nomenclature Committee report lists more than 500 different HLA class I and class II alleles [2]. HLA polymorphism tends to concentrate in hypervariable regions. Crystallographic x-ray studies [3] have demonstrated that the hypervariable regions corre- spond to MHC binding pockets which engage specific “anchor” residues of peptide ligands. Sequencing of nat- urally processed peptides co-isolated with purified MHC [4], and assays utilizing purified HLA molecules [5–7], allowed the definition of the peptide binding specificity of different HLA molecules and revealed that different MHC molecules are characterized by different ligand specificities. Epitope-based vaccines are a possible avenue to treat and prevent infectious disease and cancer. However, if specific epitopes for a large number of different MHC specificities have to be defined, the large degree of HLA polymorphism could represent a significant challenge. Studies by our group have demonstrated that different HLA class I molecules can recognize very similar peptide binding motifs, and that a significant overlap exists in their peptide binding repertoires. For example, the pep- tide binding repertoires of A*0301 and A*1101 overlap with those of A*3101, A*3301, and A*6801 [8]. A significant overlap in peptide binding repertoires has also been demonstrated among several serologically distant HLA-B alleles, including B*0702, B*3501, B*5101, From Epimmune, Inc. (J.S., S.S., M.J.N., A.S.), San Diego, CA, and the Department of Pathology (D.L.M., M.A.F.-V.), Division of Immuno- genetics, University of Maryland Medical School, Baltimore, MD, USA. Address reprint requests to: Dr. Alessandro Sette, Epimmune, Inc., 5820 Nancy Ridge Drive, Ste. 100, San Diego, CA 92121, USA; Tel: (858) 860-2516; Fax: (858) 860-2600. Received April 9, 2001; accepted July 5, 2001. Human Immunology 62, 1200 –1216 (2001) 0198-8859/01/$–see front matter © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc. PII S0198-8859(01)00319-6