DEPLETION OF THE HELPER INDUCER (MEMORY) T-CELL SUBSET USING A BISPECIFIC ANTIBODY-TOXIN CONJUGATE DIRECTED AGAINST CD4 AND CD29/

We have developed a bispecific antibody that recognizes the CD4 and CD 29 antigens simultaneously and that was examined for its ability to ta rget CD4(+)CD29(bright) T cells. The premise for using bispecific anti body-toxin conjugates was that monovalent binding to one antigen would not result in internalization while binding through both antigens wou ld predispose toward endocytosis and delivery of the toxin to the cell interior. In this study, we show that the bispecific antibody binds m onovalently to CD4 and CD29 and bivalently to both antigens. Both mono valent and bivalent binding rendered the target cells sensitive to com plement-mediated lysis, demonstrating that this effector modality cann ot take advantage of the dual specificity of the antibody. Bivalent bi nding, however, allowed modulation of more than 60% of the bound antib ody off the surface of CD4(+)CD29(bright) cells, which we further expl oited to deliver a toxin moiety preferentially to CD4(+)CD29(bright) c ells. Immunoconjugates incorporating blocked ricin (a ricin holotoxin that has its intrinsic galactose-binding sites blocked by chemically l inked affinity ligands) preferentially killed CD4(+)CD29(bright) cells in vitro by a factor of 25 in comparison with killing of total CD4(+) cells in functional assays. Assays on resting PBL demonstrated a simi lar specificity of the bispecific immunotoxin for CD4(+)CD29(bright) c ells with a concomitant relative survival of CD4(+)CD29(dim) (CD45RA()). We demonstrated that the potency of the immunotoxin is not only a function of its affinity but also of its propensity to internalize, an d that this property is influenced by the degree of bivalent binding. These results open up the possibility of engineering bispecific antibo dy toxin conjugates for use as therapeutic immunotoxins for selective removal of restricted T cell subsets.