Characterization of monoclonal antibodies specific for the V beta 3 family of the human T cell receptor generated using soluble TCR beta-chain.

A soluble, recombinant form of the human T cell receptor (TCR) beta-chain containing the V beta 3.1 sequence has been constructed, expressed in Chinese hamster ovary cells, amplified by dihydrofolate reductase selection, and purified in quantities appropriate for the generation of monoclonal antibodies (mAb). The V beta 3 sequence was chosen because of its reported elevated usage in the synovial T cells of rheumatoid arthritis patients but the approach described should be applicable to other known human V beta gene sequences. By this method, two mAb were prepared which reacted with up to 10% of normal, live peripheral blood T cells but with reactivity varying greatly among individual donors. Both mAb specifically bound to a murine T cell line transfected with a human TCR V beta 3.1 and immunoprecipitated a protein of the expected molecular weight for the TCR beta-chain. Both antibodies were mitogenic for T cells and analysis of peripheral blood lymphocyte cultures stimulated with the mAb suggested that both were specific for the V beta 3.1 subfamily and not D beta or J beta. Clones expressing V beta 3, which were derived from mAb-stimulated peripheral blood lymphocytes of a single individual, preferentially (8/13), but not exclusively, utilized the J beta 2.7 gene segment. The V beta 3.1 usage showed no preference for the CD8+ or CD4+ subpopulations of normal peripheral blood T cells.

Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.

Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.