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.
SummaryT cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (~20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDRl-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HSS) which bound B7-1 even better. Inclusion of the CDR2-1ike domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1-and CDR3-analogous regions. FLor T cells to respond to an antigenic stimulus, multiple activation signals are required from the APC (1, 2). An antigen-specific signal occurs when the TCR binds to antigenic peptides presented by MHC molecules. An important nonspecific costimulatory signal is delivered to the T cell when B7-related receptors on APCs bind to CD28 and/or CTLA-4. There are at least two homologous B7 family members found on APCs, B7-1 (also called B7 or CD80) and B7-2, both of which can deliver costimulatory signals to T cells via CD28 and/or CTLA-4 (3-7). Costimulation through CD28 or CTLA-4 is essential for T cell activation since a soluble Ig fusion protein of CTLA-4 (CTLA4Ig) blocks T cell-dependent immune responses in vitro and in vivo (5,8,9). Failure to deliver this second signal may lead to clonal inactivation or T cell anergy (10-12).CD28 and CTLA-4 both contain a single Ig V-like extracellular ligand binding domain which share •25% sequence homology (13-15). The B7 ligands are also members of the Ig superfamily (IgSF) 1 but have, in contrast to CD28 and CTLA-4, two Ig domains in their extraceUular region, an NH2-terminal V-like domain and a C-like domain (3, 6, 7). CTLA4Ig binds to B7-1 with "~20 fold higher avidity tha...
The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99YPPPY104 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.
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 GFCCC -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 -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 GFCC face of the IgV domain and the ABED face of the IgC domain.Distinct activation signals from antigen-presenting cells are required for T lymphocyte-dependent immune responses (1-3). Antigen-specific activation of T cells is mediated by peptide/ major histocompatibility complexes on antigen-presenting cells interacting with specific T cell antigen receptors. Binding of CD28 and/or CTLA-4 on T cells to B7-related receptors on antigen-presenting cells provides important antigen-nonspecific costimulatory signals essential for optimum immune responses. Blocking the delivery of these costimulatory signals in vitro can lead to T cell unresponsiveness or anergy (4 -6). Two B7-related molecules, each having different patterns of expression, have been identified: CD80 (B7-1) (7, 8) and CD86 (B7-2/ B70) (9 -11).CD80 and CD86 are members of the immunoglobulin superfamily (IgSF), 1 with their extracellular regions consisting of one amino-terminal Ig variable-like (IgV) and one membrane proximal Ig constant-like (IgC) domain. The B7 IgV domains may include some structural features that depart from currently known Ig folds (12) and share sequence similarity with three major histocompatibility complex-encoded members of the IgSF (13). In contrast, the IgC domains display significant sequence-structure compatibility with  2 -microglobulin (12). B7 molecules are not disulfide-linked, unlike their coreceptors CTLA-4 and CD28, which are also IgSF members (14 -16).Despite having only ϳ25% sequence h...
When human granulocytes were exposed to 50 nM N-formyl-Met-Leu-[ 3 H]Phe at 37°C they rapidly formed ligand-receptor complexes that dissociated 50-100 times more slowly than those on cells initially exposed to the peptide at 4°C . These complexes of apparent higher affinity were stable after detergent solubilization of the cells with Triton X-100. The complexes co-isolated with the detergent insoluble cytoskeletal residues and were free of the cytosolic and Golgi markers, lactate dehydrogenase and galactosyl transferase, respectively .After 5 s of exposure to f-Met-Leu-Phe, 2,000-3,000 molecules of ligand per cell were trapped in such complexes. Continued exposure resulted in capture of a maximum of 14,000 molecules per cell by 5 min . Exposure at 15°C, a temperature at which endocytosis of the receptor is prevented, resulted in complex formation at a linear rate for at least 20 min to levels twice those measured at 37°C . At 4°C, complex formation was -10% of the maximum amount formed at 37°C . Pulse-chase experiments revealed that the complex was in transient association with the cytoskeleton with a half life ranging between 30 s to 4 min depending on the length of the original incubation . Electron microscopic autoradiography indicated that after 1 min of incubation at 37°C, the majority of the specific autoradiographic grains were localized to the outer circumference of the cellular cytoskeleton . After 4 min of incubation, the grains were less frequent at the cytoskeleton periphery but still threefold enriched over a random cellular distribution . We conclude that a metabolically controlled modulation of the state of the N-formyl chemotactic peptide receptor occurs in the plasma membrane which may be the result of transient association of ligand-receptor complex and the cell cytoskeleton .
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