VISTA is an acidic pH-selective ligand for PSGL-1.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Discovery of potent and selective nonsteroidal indazolyl amide glucocorticoid receptor agonists.

Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure.

Involvement of DPP-IV catalytic residues in enzyme-saxagliptin complex formation.

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

The structure of the death receptor 4-TNF-related apoptosis-inducing ligand (DR4-TRAIL) complex.

The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3 Šresolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.

Development and characterization of antibody reagents to assess anti-PEG IgG antibodies in clinical samples.

BACKGROUND: Polyethylene glycol (PEG) is a polymer that can be conjugated with therapeutic proteins. Monitoring anti-PEG antibodies in human subjects may be required as part of immunogenicity assessment. The lack of well-characterized anti-PEG reagents have limited our understanding of anti-PEG humoral response. RESULTS: Antibodies reactive to PEG were engineered with a human IgG1 Fc. Surface plasmon resonance and plate-based methods demonstrated that their binding was dependent on molecular weight (MW) of PEG. Specificity experiments using chemical analogs identified their specificity. CONCLUSION: Affinity, specificity and MW of PEG are critical characteristics that impact interactions of anti-PEG antibodies with PEG. These attributes especially MW of PEG and the assay formats may impact the ability to detect anti-PEG antibodies.

Comparative studies of active site-ligand interactions among various recombinant constructs of human beta-amyloid precursor protein cleaving enzyme.

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.