Biomedical subjects

Robert F Kelley

Publications and source records attributed to Robert F Kelley.

8 recordsLinked to original sources

PubMed2006

Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.

Amino Acid Sequence↗

PubMed2005

Tyrosine plays a dominant functional role in the paratope of a synthetic antibody derived from a four amino acid code.

The antigen-binding fragment Fab-YADS2 recognizes vascular endothelial growth factor (VEGF) and was derived from a library with chemical diversity restricted to only four amino acids (Tyr, Ser, Ala and Asp). The structure of the Fab:antigen complex revealed that the structural paratope is dominated by Tyr side-chains. Isothermal titration calorimetry and cell-based assays show that restricted chemical diversity does not limit the affinity or specificity of Fab-YADS2, which behaves in a manner comparable to natural antibodies. Mutagenesis experiments reveal that the functional paratope is dominated by Tyr, which represents 11 of the 15 functionally important residues. However, mutagenesis experiments also indicate that substitution of any of these tyrosine residues by Phe does not significantly affect binding to VEGF. Furthermore, saturation mutagenesis shows that replacement of three functionally important tyrosine residues by combinations of other hydrophobic residues is not only tolerated, but can actually improve affinity. The results support a model for naïve antigen recognition in which large Tyr side-chains establish binding contacts with antigen, and small Ser and Ala side-chains serve as auxiliaries that help to position Tyr in favorable binding conformations. While Tyr may not be optimal for any particular antigen contact, it is nonetheless capable of mediating favorable interactions with a diverse array of surfaces. Furthermore, the side-chain hydroxyl group makes Tyr significantly more hydrophilic than Phe and other hydrophobic amino acids. Increased hydrophilicity may reduce non-specific binding in the unbound state, and this may be critical for a naïve repertoire that is exposed to a diverse range of potential antigenic surfaces. The results show that the chemical nature of Tyr endows the amino acid with a privileged role in antigen recognition, and this likely explains the high abundance of Tyr in natural antigen-binding sites.

Amino Acid Sequence↗

PubMed2004

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding.

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.

Alternative Splicing↗

PubMed2004

Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling.

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors.

Apoptosis↗

PubMed2004

Similar molecular interactions of factor VII and factor VIIa with the tissue factor region that allosterically regulates enzyme activity.

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.

Allosteric Regulation↗

PubMed2004

Engineering an APRIL-specific B cell maturation antigen.

B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.

Amino Acid Substitution↗

PubMed2003

BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site.

BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs). However, the extracellular domain (ECD) of BR3 consists of only a partial CRD, with cysteine spacing distinct from other modules described previously. Herein, we report the solution structure of the BR3 ECD. A core region of only 19 residues adopts a stable structure in solution. The BR3 fold is analogous to the first half of a canonical TNFR CRD but is stabilized by an additional noncanonical disulfide bond. BAFF-binding determinants were identified by shotgun alanine-scanning mutagenesis of the BR3 ECD expressed on phage. Several of the key BAFF-binding residues are presented from a beta-turn that we have shown previously to be sufficient for ligand binding when transferred to a structured beta-hairpin scaffold [Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M., Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., Starovasnik, M. A, and Dixit, V. M. (2002) Immunity 10, 515-524]. Outside of the turn, mutagenesis identifies additional hydrophobic contacts that enhance the BAFF-BR3 interaction. The crystal structure of the minimal hairpin peptide, bhpBR3, in complex with BAFF reveals intimate packing of the six-residue BR3 turn into a cavity on the ligand surface. Thus, BR3 binds BAFF through a highly focused interaction site, unprecedented in the TNFR family.

Amino Acid Sequence↗

PubMed2002

Enhancing the anticoagulant potency of soluble tissue factor mutants by increasing their affinity to factor VIIa.

Complexation of factor VIIa (FVIIa) and tissue factor (TF) initiates the extrinsic pathway of blood coagulation. Inappropriate triggering of this pathway has been linked to thrombotic disorders. We have previously shown that a mutant form ofsoluble tissue factor (sTF, residues 1-219) having Lys165 and Lys166 replaced with alanine residues (hTFAA) gave an antithromboticeffect in a rabbit model of arterial thrombosis. Here we have improved the potency of hTFAA by incorporating amino acid substitutions thatincrease the affinity of sTF for FVIIa. Phage display has been used toselect consensus sequences at two FVIIa-contact regions on sTF, segments 44-50 and 130-140, that individually showed modestly increased (approximately 2-fold) FVIIa-affinity. These consensus sequences have been combined with other previously selected mutations to generate a series of variants (hTFAA-2, hTFAA-3, and hTFAA-4) having 4 to 10-fold increased FVIIa-binding affinity. Surface plasmon resonance measurementsindicate that the increased affinity results mostly from an increase in the association rate constant. All of these variants displayed increased inhibitor potency relative to hTFAA in an assay of Factor X activation catalyzed by the complex of FVIIa with membrane TF. In addition, these affinity improved hTFAA variants are more potent anticoagulants and have increased antithrombotic activity relative to hTFAA in an ex vivo flow chamber model of thrombosis. The potency trend parallels increases in FVIIa-binding affinity; however, the absolute fold increases in potencies are greater than increases in binding affinity, consistent with kinetic studies of the FVIIa-binding event. Through incorporation of 10 amino acid substitutions (hTFAA-3) we have increased the antithrombotic activity of hTFAA by 20-fold.

Anticoagulants↗