MAA868, a novel FXI antibody with a unique binding mode, shows durable effects on markers of anticoagulation in humans.

A large unmet medical need exists for safer antithrombotic drugs because all currently approved anticoagulant agents interfere with hemostasis, leading to an increased risk of bleeding. Genetic and pharmacologic evidence in humans and animals suggests that reducing factor XI (FXI) levels has the potential to effectively prevent and treat thrombosis with a minimal risk of bleeding. We generated a fully human antibody (MAA868) that binds the catalytic domain of both FXI (zymogen) and activated FXI. Our structural studies show that MAA868 traps FXI and activated FXI in an inactive, zymogen-like conformation, explaining its equally high binding affinity for both forms of the enzyme. This binding mode allows the enzyme to be neutralized before entering the coagulation process, revealing a particularly attractive anticoagulant profile of the antibody. MAA868 exhibited favorable anticoagulant activity in mice with a dose-dependent protection from carotid occlusion in a ferric chloride-induced thrombosis model. MAA868 also caused robust and sustained anticoagulant activity in cynomolgus monkeys as assessed by activated partial thromboplastin time without any evidence of bleeding. Based on these preclinical findings, we conducted a first-in-human study in healthy subjects and showed that single subcutaneous doses of MAA868 were safe and well tolerated. MAA868 resulted in dose- and time-dependent robust and sustained prolongation of activated partial thromboplastin time and FXI suppression for up to 4 weeks or longer, supporting further clinical investigation as a potential once-monthly subcutaneous anticoagulant therapy.

Characterization of the in vitro and in vivo properties of CFZ533, a blocking and non-depleting anti-CD40 monoclonal antibody.

The CD40-CD154 costimulatory pathway is essential for T cell-dependent immune responses, development of humoral memory, and antigen presenting cell function. These immune functions have been implicated in the pathology of multiple autoimmune diseases as well as allograft rejection. We have generated CFZ533, a fully human, pathway blocking anti-CD40 monoclonal antibody that has been modified with a N297A mutation to render it unable to mediate Fcγ-dependent effector functions. CFZ533 inhibited CD154-induced activation of human leukocytes in vitro, but failed to induce human leukocyte activation. Additionally, CFZ533 was unable to mediate depletion of human CD40 expressing B cells. In vivo, CFZ533 blocked primary and recall T cell-dependent antibody responses in nonhuman primates and abrogated germinal formation without depleting peripheral blood B cells. We also established a relationship between plasma concentrations of CFZ533 and CD40 pathway-relevant pharmacodynamic effects in tissue. Collectively these data support the scientific rationale and posology for clinical utility of this antibody in select autoimmune diseases and solid organ transplantation.

A combined approach to early detect in vitro drug-induced hemostatic changes in preclinical safety.

Early detection of drug-induced alterations of hemostasis is challenging. Drugs can affect different components of the Virchow's triad and measurement of plasmatic coagulation times lacks sensitivity. New techniques for a more global assessment of the hemostasis are now available: the impedance platelet aggregometry, the thromboelastography and the thrombin generation measurement. The aim of this study was to evaluate three techniques (i.e.: Multiplate®, TEG® and CAT) for the in vitro detection of the effect of a drug known to induce hemostatic alterations in a preclinical safety environment. Cyclosporine A was chosen and tested at 4 concentrations after solubilization in DMSO in Wistar rats and Beagle dogs. The results obtained were comparable between both species except for the thrombin generation in platelet rich plasma. Enhanced platelet aggregability was observed after ADP stimulation and alterations of the thromboelastograms consisted in decreased maximum amplitude and increased LY30. A dual effect on thrombin generation was observed and suggested that CsA may interact with platelets in rat platelet rich plasma and speed up thrombin generation. The results of this study indicate that using a combined approach on hemostasis testing in preclinical safety it is possible to detect in vitro drug-induced alterations of hemostasis.

Reduced Activity of Sphingosine-1-Phosphate Lyase Induces Podocyte-related Glomerular Proteinuria, Skin Irritation, and Platelet Activation.

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction.

Optimization of Multiplate(®) whole blood platelet aggregometry in the Beagle dog and Wistar rat for ex vivo drug toxicity testing.

This study was performed to optimize and standardize the use of the Multiplate(®) whole blood impedance aggregometer in the Beagle dog and Wistar rat for use in a research laboratory environment. The anticoagulants citrate, heparin and hirudin were compared and platelet aggregation responses to ADP, collagen, arachidonic acid and Par-4 agonist were evaluated to determine their half maximal effective concentrations (EC(50)) in blood containing low concentrations of a drug solvent (0.1% DMSO). The results indicate that citrate anticoagulation is not suitable for Multiplate(®) whole blood aggregometry because of the presence of spontaneous aggregation. ADP and collagen were found to be appropriate agonists for both species, whereas in the Beagle dog Par-4 agonist failed to induce aggregation and arachidonic acid induced platelet aggregation showed a high interindividual variability. The agonists EC(50) calculated in hirudin blood were 2.70 µM ADP, 0.85 µg/ml collagen, 0.03 mM arachidonic acid and 165.7 µM Par-4 agonist in the Wistar rat, and 0.95 µM ADP and 0.23 µg/ml collagen in the Beagle dog.

Large-scale prediction and testing of drug activity on side-effect targets.

Discovering the unintended 'off-targets' that predict adverse drug reactions is daunting by empirical methods alone. Drugs can act on several protein targets, some of which can be unrelated by conventional molecular metrics, and hundreds of proteins have been implicated in side effects. Here we use a computational strategy to predict the activity of 656 marketed drugs on 73 unintended 'side-effect' targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 µM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a drug-target-adverse drug reaction network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic oestrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme cyclooxygenase-1. The clinical relevance of this inhibition was borne out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.

Immunogenicity and tolerability of an inactivated and adjuvanted pandemic H1N1 influenza vaccine, in HIV-1-infected patients.

We evaluated the efficacy and tolerability of a single dose of the split virion AS03-adjuvanted pandemic H1N1 influenza vaccine (A/California/7/2009) in 84 HIV-1 infected individuals. Antibody titers were determined by hemagglutination inhibition assay and by microneutralization. Vaccine was well tolerated. At 21 days post vaccination, 56 (67%) patients had seroconverted. There was no correlation between baseline CD4 cell count (p=0.539) or HIV viral load (p=0.381) and immune response. Other vaccine strategies should be evaluated in this HIV population, to improve response rates.

CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro.

INTRODUCTION: Aberrant signaling within and between B and T cells, considered to be central in systemic lupus erythematosus (SLE), could depend on enhanced CD40-CD154 activation. As a result, autoreactive B cells, normally anergic, differentiate and secrete antibodies attacking several normal tissues. Thus restorating B cell homeostasis might help control this disease. In this study, two facets of SLE B cells were investigated, namely their in vitro response to CD40-CD154 and the effect of treatment with human immunoglobulins for intravenous use (IVIg). MATERIALS AND METHODS: Blood samples from SLE patients and healthy volunteers were obtained and used to isolate B cells, which were activated through CD40 in the presence or absence of IVIg. The phenotype, proliferation, and differentiation of the SLE B cells were determined and compared with those of control B cells using flow cytometry and standard ELISA. RESULTS: In this model, CD40-activated SLE B cells, as control B cells, proliferated and differentiated and were characterized by the emergence of CD19(lo)CD38(++)CD138(+)CD27(++) cells. IVIg treatment of the CD40-activated SLE B cells resulted in higher differentiation, characterized by increased secretion rates of IgG and IgM, as reported previously for control B cells. CONCLUSIONS: Taken as a whole, such accelerated differentiation of CD40-activated B cells suggests that IVIg may participate in re-equilibration of the antibody repertoire by replacing pathological antibodies by de novo harmless antibodies.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Human B lymphocytes and non-Hodgkin's lymphoma cells become polyploid in response to the protein kinase inhibitor SU6656.

We show that prolonged exposure of non-Hodgkin's lymphoma (NHL) cell lines to low doses of the Src family protein tyrosine kinases (SFKs) inhibitor SU6656 caused proliferation abrogation as a result of the formation of cells with single multilobed nuclei and several mitotic spindle poles, features similar to polyploid megakaryocytes. The propensity of the NHL B cells tested to undergo polyploid was unrelated to the presence of p53 mutations in these cells since comparable outcomes were observed in SU6656-exposed cultures of blood B lymphocytes derived from healthy individuals. Thus, in addition to its utility for the study of megakaryocyte polyploidization, our results show that SU6656 can also induce polyploidy in cells of lymphoid origin, revealing a chemotherapeutic potential for this inhibitor to limit tumor propagation of malignant B cell lymphomas, although not without affecting normal B cells as well.

Comparison of promoter activities for efficient expression into human B cells and haematopoietic progenitors with adenovirus Ad5/F35.

Adenoviral gene transfer into human B lymphocytes and haematopoietic progenitors would allow the characterization of their function on cellular growth, differentiation and survival. Efficient gene expression is however strongly dependent on the promoter used. In this study, we investigated the relative strength of various promoters by following and measuring the expression of the reporter gene EYFP in human peripheral B lymphocytes, cord blood CD34(+) cells and the megakaryocytic cell line M-07e. The murine PGK promoter provided the best level of transgene expression in CD34(+) cells among the four promoters tested, followed closely by the CMV promoter, and to a lesser extend by a CMV promoter with a beta-globin/IgG chimeric intron, whereas the human CD40 promoter provided the lowest levels of expression. In contrast, the strongest promoters in B lymphocytes were the two CMV promoters. Surprisingly, even the best promoters were unable to induce transgene expression in more than 75-80% of the primary B and CD34(+) cells, even though 100% of the cells were infected. Finally and in contrast to retroviruses, only a minority of B lymphocytes and CD34(+) cells were able to induce the transcription of IRES-containing bicistronic expression cassettes from adenovirus.

The survival of IL-6-dependent myeloma cells critically relies on their capability to transit the G1 to S phase interval of the cell cycle.

Interleukin-6 (IL-6) has an essential role in the initial progression of myeloma cell tumours. IL-6 triggers proliferation of these cells via the Ras-mitogen-activated protein kinase (MAPK) cascade and is thought to promote their survival via signal transducer and activator of transcription (STAT) pathway-dependent regulation of Bcl-2 family antiapoptotic members. Using IL-6-dependent murine B9 hybridoma/plasmacytoma cells, we here report that exiting the cell cycle G1 phase is a crucial step contributing to maintain viability. We show that (1) drug-mediated reversible G1 arrest triggered apoptosis despite the presence of IL-6; (2) a short IL-6 pulse to G1-arrested cells was sufficient to induce S phase entry and prevent apoptosis; and (3) phorbol ester and related derivatives promoted S phase entry and survival of IL-6-starved cells without up-regulating bcl-XL expression. Furthermore, that the MAPK kinase (MEK) 1/2 inhibitor, U0126, blocked proliferation and induced death of B9 cells indicate that IL-6 may not exert its survival effect primarily through bcl-XL and emphasizes the key role of Ras-MAPK cascade elements in the regulation of myeloma growth/viability.

Regulation of growth-related genes by interleukin-6 in murine myeloma cells.

Interleukin-6 (IL-6), a pleiotropic cytokine with effects on several hematopoietic and other normal cells, is also important for the growth and survival of tumor cells such as murine plasmacytomas and human myelomas. Exploiting the 11A3 hybridoma cell line for its IL-6 requirement to proliferate in vitro, we used subtractive suppression hybridization (SSH) to identify genes whose expression is stimulated and/or repressed in response to IL-6. Northern blot analysis of 100 arbitrarily picked subtracted cDNA clones revealed that expression of 11 mRNAs were IL-6-modulated. Among these, eight were genes known to encode a variety of proteins such as enzymes (PCK, MTDNI), structural proteins (Tropoelastin), transcriptional regulators (BRG1) and proteins involved in cell division control (Cyclin A, OAZi) or cell signaling (PIX, TOPK/PBK). The recently identified MAPKK-like protein kinase TOPK/PBK gene represents a likely candidate IL-6 target gene as suggested by its significant up-regulated expression in hybridoma cells induced to grow by a brief IL-6 pulse. The diversity of growth-related genes identified in this study further emphasizes the central role of IL-6 in the regulation of myeloma cell expansion in addition to its previously demonstrated role in the inhibition of apoptosis.

Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures.

B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.

Expression of gooseberry-proximal in the Drosophila developing nervous system responds to cues provided by segment polarity genes.

Segment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis.