ABSTRACT. Diallelic polymorphisms have been identified in the HLA-H gene and the ZNF192 gene located about 2 megabases centromeric to HLA-H. The three polymorphic sites in HLA-H together with the two hemochromatosis mutations in this gene give rise to 8 different haplotypes. The three polymorphic sites in ZNF192 give rise to 4 different haplotypes. The haplotypes in HLA-H are in complete linkage disequilibrium with the two common mutations in that gene, 845A (C282Y) and 187G (H63D). The 845A mutation is in weak linkage disequilibrium with the ZNF192 polymorphisms and the 187G mutation appears to be in equilibrium with this polymorphism. The 187G mutation therefore appears to be the older of the two HLA-H mutations.

Keywords: HLA-H, hemochromatosis, linkage, mutation.

Reprint requests to: Ernest Beutler, M.D., The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 North Torrey Pines Road, La Jolla, CA 92037, phone: (619) 784-8040, fax: (619) 784-2083, e-mail: beutler@scripps.edu.

ABSTRACT. Two antigenized antibodies (AgAbs) were engineered to express peptidic Arg-Gly-Asp (RGD) motifs present in extracellular matrix molecules. The RGD tripeptide sequence was inserted in the third hypervariable loop of an immunoglobulin human/mouse chimeric heavy chain gene as a single or three repeat yielding two antibodies termed gamma1RGD and gamma1(RGD)3, respectively. The antibodies were used to target specific cell-surface receptors of the integrin type expressed by three human tumor cell lines, a melanoma (M21), an osteosarcoma (KRIB) and a fibroblastoma (WI-38). Based on in vitro adhesion assays and flow cytometric analysis, we found that all three cell lines interacted with gamma1(RGD)3 but not with gamma1RGD. Binding of tumor cells to surface-immobilized gamma1(RGD)3 was inhibited in a dose-dependent manner by the RGD-containing synthetic peptides GdRGDSP and RGDS. These synthetic peptides, but not a GDR- containing control peptide, interfered with the binding of tumor cells to surface-immobilized human fibronectin. In their soluble form, neither fibronectin nor gamma1(RGD)3 inhibited tumor cell adhesion to surface-immobilized fibronectin. Gamma1(RGD)3 specifically recognized integrin alpha v beta 3 based on two criteria: reactivity with purified integrin receptors and binding to variants of M21 melanoma cells expressing alpha v beta 3, alphaIIbbeta3 or no beta3 integrins, respectively. Collectively, our results indicate that the (RGD)3 loop in the antigenized antibody mimics the ligand function of natural extracellular matrix proteins and has a restricted receptor specificity for the alpha v beta 3 integrin which is not inherent to short RGD containing peptides.

Keywords: RGD, antibody, antigenized, alpha v beta 3, integrin, adhesion, tumor cells, melanoma-M21, osteosarcoma-KRIB, fibroblastoma-WI-38.

Reprint requests to: Maurizio Zanetti, M.D., Department of Medicine and Cancer Center, University of California, San Diego, 9500 Gilman Drive, 0063, La Jolla, CA 92093-0063, phone: (619) 534-7217, fax: (619) 534-5792, e-mail: mzanetti@UCSD.edu.