Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists

Lühder F, Huang Y, Dennehy KM, Guntermann C, Müller I, Winkler E, Kerkau T, Ikemizu S, Davis SJ, Hanke T, Hünig T. (2003), J Exp Med. 197, 955-66

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.

Key figure: Depiction of the epitopes for superagonistic and conventional CD28-specific mAbs in a three-dimensional model of the extracellular part of human CD28

The model of the CD28 monomer was derived by computer calculation from the X ray crystallographic structure of murine CTLA-4 using the sequence alignment derived by Metzler et al. (reference 25). The dimer was constructed on the basis of the CTLA-4 homodimer observed in the crystals of the complex of CTLA-4 and CD80, and is shown with the membrane-proximal region at the top. The MYPPPY motif (aa 99–104) critical for B7 binding is indicated in green, the adjacent aa 98 residue critical for binding of the conventional rat and mouse CD28-specific mAb is highlighted in yellow, and the C′′D loop responsible for the binding of superagonistic rat and human CD28-specific mAb (aa 60–65) is indicated in red.