Joseph Roy Naemura

Rational Development of LEA29Y (belatacept), a High‐Affinity Variant of CTLA4‐Ig with Potent Immunosuppressive Properties

Current success in organ transplantation is dependent upon the use of calcineurin‐inhibitor‐based immunosuppressive regimens. Unfortunately, current immunotherapy targets molecules with ubiquitous expression resulting in devastating non‐immune side effects. T‐cell costimulation has been identified as a new potential immunosuppressive target. The best characterized pathway includes CD28, its homologue CTLA4 and their ligands CD80 and CD86. While an immunoglobulin fusion protein construct of CTLA4 suppressed rejection in rodents, it lacked efficacy in primate transplant models. In an attempt to increase the biologic potency of the parent molecule a novel, modified version of CTLA4‐Ig, LEA29Y (belatacept), was constructed. Two amino acid substitutions (L104E and A29Y) gave rise to slower dissociation rates for both CD86 and CD80. The increased avidity resulted in a 10‐fold increase in potency in vitro and significant prolongation of renal allograft survival in a pre‐clinical primate model. The use of immunoselective biologics may provide effective maintenance immunosuppression while avoiding the collateral toxicities associated with conventional immunsuppressants.

Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99 YPPPY104 motif are adjacent to a patch of charged surface residues on the A‘GFCC’ face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.

Both Extracellular Immunoglobin-like Domains of CD80 Contain Residues Critical for Binding T Cell Surface Receptors CTLA-4 and CD28

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC′C″ β-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED β-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC′C″ face of the IgV domain and the ABED face of the IgC domain.

Abrogation of the antiproliferative activity of oncostatin M by a monoclonal antibody

Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.