title: Enhanced transmembrane signalling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface.
abstract: Signal transduction occurs through multiple receptors expressed on mature, resting T cells. In addition to the CD3-T cell receptor complex, the CD2, CD4, CD5, CD7, CD8 and CD28 receptors mobilize cytoplasmic calcium within minutes of binding with monoclonal antibodies and additional crosslinking occurs on the cell surface. As an approach to study the interactions between these receptors and their transduced signals, monoclonal antibodies to each of these receptors were covalently coupled as heteroconjugates and investigated for activity in cytoplasmic calcium mobilization using indo-1 and flow cytometry. Of a total of 35 conjugates studied, there were seven heteroconjugates that showed an increase in activity and these consisted of either certain conjugates of anti-CD3 or certain conjugates of anti-CD5. The CD3-CD2, CD3-CD4, CD3-CD6 and CD3-CD8 heteroconjugates each gained two to three orders of magnitude in titer in calcium mobilization compared to unconjugated CD3 or the CD3-CD3 conjugate. The increase in activity was not accompanied by an increase in binding titer, indicating that signal transduction occurred at lower levels of receptor occupancy. The increased activity was dependent in each case on the relevant second receptor, since unconjugated CD2, CD4, CD6 or CD8 MAb could block the activity of the corresponding heteroconjugate. Neither CD3-CD5, CD3-CD28 or CD3-CD3 conjugates gained activity, whereas CD3-CD7 heteroconjugates gained slightly in activity. The heteroconjugates with CD5 that acquired ability to mobilize calcium at low concns (less than 5 micrograms/ml) were CD5-CD4, CD5-CD8 and CD5-CD6. Their activity could be inhibited by either CD5 MAb or the second MAb of the heteroconjugate. The increased activity of CD3 or CD5 heteroconjugates was observed in the absence of extracellular calcium. Size exclusion chromatography of heteroconjugates demonstrated that 1:1 ratios were optimal, but larger conjugates were also active. These results suggest that certain receptors are capable for molecular interactions on the cell surface to form complexes with enhanced activity in signal transduction leading to calcium mobilization.
authors.affiliation: Oncogen, Seattle, WA 98121.
authors.forename: J A
authors.initials: JA
authors.lastname: Ledbetter
authors.forename: N A
authors.initials: NA
authors.lastname: Norris
authors.forename: A
authors.initials: A
authors.lastname: Grossmann
authors.forename: L S
authors.initials: LS
authors.lastname: Grosmaire
authors.forename: C H
authors.initials: CH
authors.lastname: June
authors.forename: F M
authors.initials: FM
authors.lastname: Uckun
authors.forename: W L
authors.initials: WL
authors.lastname: Cosand
authors.forename: P S
authors.initials: PS
authors.lastname: Rabinovitch
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D002118:Calcium