Human single-chain variable fragment that specifically targets arthritic cartilage.

OBJECTIVE: To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. METHODS: We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti-ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti-ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti-ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II-Fc fusion protein (mTNFRII-Fc) was also investigated. RESULTS: The anti-ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti-ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. CONCLUSION: Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis.

Gene structure and promoter functional analysis of the marmoset type II GnRH receptor.

The marmoset type II GnRH receptor (GnRH-R) gene has the same structure and genomic organisation as the human and other type II GnRH-R genes. The gene consists of three exons and two introns and overlaps in the antisense orientation on its 5' end with peroxisomal membrane protein 11beta and on its 3' end with the RNA-binding motif protein 8A. However, these genes occur only at one locus in the marmoset genome, while in the human at two loci. Employing 5' rapid amplification of cDNA ends demonstrated that the marmoset type II GnRH-R gene has two transcriptional start sites at -341 and -567 nucleotides relative to the translational start codon and both start sites lack TATA and CAAT consensus sequences. A luciferase reporter construct with a 2.3 kb 5' flanking region of the type II GnRH-R gene was active in a wide variety of cell lines tested, consistent with the wide tissue expression of the receptor. Progressive 5' and 3' deletions were employed to identify sequences required for basal expression of the type II GnRH-R gene. This analysis identified negative regulatory elements in the regions -2342/-1995, -1679/-1084 and -458/-1 and positive regulatory elements in the regions -1995/-1679, -1084/-458 and -458/-1 relative to the translational start site. The strongest of the positive regions located between -766/-665 has enhancer activity when cloned in front of a heterologous minimal promoter and is critical for basal expression of the type II GnRH-R.