Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

A rate-limiting role for Dickkopf-1 in bone formation and the remediation of bone loss in mouse and primate models of postmenopausal osteoporosis by an experimental therapeutic antibody.

Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays.

Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

Comparative chemical attributes of native North American hop, Humulus lupulus var. lupuloides E. Small.

The genetic diversity of 159 representative genotypes of native hop (Humulus lupulus var. lupuloides E. Small, Cannabaceae) from 34 selected populations was assessed by relative magnitudes and ranges of alpha acids (AA), beta acids (BA), and the cohumulone (CoH) component of alpha acids, with reference to temporal changes between 1989-1990 and 2001, and to the same attributes in American and European hop cultivars, principally H. lupulus var. lupulus L. Chemical profiles of these genotypes were generated by high pressure liquid chromatography (HPLC) of methanol extracts from their processed samples (cones). The alpha ratio (AR, alpha acids / alpha+beta acids) measured the degree to which alpha acids predominated in cone extracts. Synchronous ranges of AR and CoH were also selected for graphic portrayals of native hop genotypic diversity. Cones sampled and analyzed from eight populations that were accessible in both 1989 and 2001 were distinct in chemical attributes, indicating a succession of genotypes, and suggesting temporal cycling of H. lupulus var.lupuloides germplasm. The principal distinctions between the two sub-species were a markedly higher proportion of CoH (38-88% vs. 19-41%) in alpha acids of H. l. var. lupuloides, and generally higher concentrations of AA in cultivars of both American and European commercial hop cultivars, predominantly H. lupulus var. lupulus. All of the 159 native hop genotypes also contained detectable levels of xanthohumol and xanthogalenol, prenylflavonoids recently reported to have mammalian anti-cancer activity. Some native genotypes had previously exhibited natural repellence of insect and mite pests; thus H. lupulus var. lupuloides germplasm offers a diverse resource of underutilized and yet undefined biochemicals.