ARTHRITIS & RHEUMATISM Vol. 46, No. 8, August 2002, pp 2109–2120 DOI 10.1002/art.10464 © 2002, American College of Rheumatology Identification of Synovium-Specific Homing Peptides by In Vivo Phage Display Selection Lewis Lee, 1 Christopher Buckley, 2 Mark C. Blades, 1 Gabriel Panayi, 1 Andrew J. T. George, 3 and Costantino Pitzalis 1 Objective. To identify homing peptides specific for human synovium that could be used as targeting devices for delivering therapeutic/diagnostic agents to human joints. Methods. Human synovium and skin were trans- planted into SCID mice. A disulfide-constrained 7–amino acid peptide phage display library was injected intravenously into the animals and synovial homing phage recovered from synovial grafts. Following 3–4 cycles of enrichment, DNA sequencing of homing phage clones allowed the identification of specific peptides that were synthesized by a-fluorenylmethyloxycarbonyl chemistry and used in competitive in vivo assays and immunohistochemistry analyses. Results. We isolated synovial homing phages dis- playing specific peptides that distinctively bound to synovial but not skin or mouse microvascular endothe- lium (MVE). They retained their tissue homing speci- ficity in vivo, independently from the phage component, the original pathology of the transplanted tissue, and the degree of human/murine graft vascularization. One such peptide (CKSTHDRLC) maintained synovial hom- ing specificity both when presented by the phage and as a free synthetic peptide. The synthetic peptide also competed with and inhibited in vivo the binding of the parent phage to the cognate synovial MVE ligand. Conclusion. This is the first report describing peptides with homing properties specific for human synovial MVE. This was demonstrated using a novel approach targeting human tissues, transplanted into SCID mice, directly by in vivo phage display selection. The identification of such peptides opens the possibility of using these sequences to construct joint-specific drug delivery systems that may have considerable impact in the treatment of arthritic conditions. The microvascular endothelium (MVE) plays a major role in the pathogenesis of rheumatoid arthritis (RA), making it an important therapeutic target. RA is a condition characterized by a proliferative synovitis that is responsible for cartilage and bone damage leading to progressive joint destruction (1,2). Florid sprouting of new blood vessels (neoangiogenesis) is typically seen in the early phases of RA synovitis, suggesting that it is a critical element in this condition (3). In the established chronic phase of the disease, the MVE is also important, since it functions as a conduit for the continuous influx of inflammatory cells from the bloodstream into the joint (4,5). The extravasation process is a complex phenom- enon regulated by a series of integrated adhesion and signaling events that include the interaction of surface cell adhesion molecules (CAMs) and chemokines (6,7). In addition to the general mechanisms applicable to all leukocyte types, there is evidence that the specific pairing of “homing receptors” and “vascular ad- dressins,” expressed on the surface of migrating lympho- cytes and on MVE of different organs, respectively, contributes to the selective recruitment of different leukocyte populations to various tissues (8,9). Well- characterized examples include the preferential interac- tion of L-selectin with glycosylation-dependent cell ad- Supported by The Special Trustees of Guy’s Hospital (PhD Studentship to Lewis Lee), The Medical Research Council (Senior Fellowship to Dr. Buckley), The Arthritis Research Campaign (Project Grant to Mark C. Blades, Endowed Chair to Dr. Panayi), and The Wellcome Trust (Senior Fellowship to Dr. Pitzalis and Project Grant to Dr. George). 1 Lewis Lee, MSc, Mark C. Blades, MSc, Gabriel Panayi, DSc, MD, FRCP, Costantino Pitzalis, MD, PhD, FRCP: Guy’s, King’s, and St Thomas’ School of Medicine, London, UK; 2 Christopher Buckley, MD, PhD: University of Birmingham, Birmingham, UK; 3 Andrew J. T. George, MA, PhD: Imperial College, Hammersmith Hospital, Lon- don, UK. Address correspondence and reprint requests to Costantino Pitzalis, MD, PhD, FRCP, Rheumatology Unit, 5th Floor, Thomas Guy House, Guy’s Hospital, London SE1 9RT, UK. E-mail: costantino.pitzalis@kcl.ac.uk. Submitted for publication February 1, 2002; accepted in revised form April 30, 2002. 2109