title: Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists.
abstract: Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): "conventional," TCR signaling-dependent costimulatory mAbs and "superagonistic" mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C"D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C"D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C"D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor kappaB pathway without inducing phosphorylation of either TCRzeta or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, D-97078 Würzburg, Germany.
authors.forename: Fred
authors.initials: F
authors.lastname: Lühder
authors.forename: Yun
authors.initials: Y
authors.lastname: Huang
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Dennehy
authors.forename: Christine
authors.initials: C
authors.lastname: Guntermann
authors.forename: Ingrid
authors.initials: I
authors.lastname: Müller
authors.forename: Erna
authors.initials: E
authors.lastname: Winkler
authors.forename: Thomas
authors.initials: T
authors.lastname: Kerkau
authors.forename: Shinji
authors.initials: S
authors.lastname: Ikemizu
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
authors.forename: Thomas
authors.initials: T
authors.lastname: Hanke
authors.forename: Thomas
authors.initials: T
authors.lastname: Hünig
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes; D006801:Humans; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016328:NF-kappa B / Q000378:metabolism; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D000939:Epitopes; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse