Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

A subcutaneous cellular implant for passive immunization against amyloid-ß reduces brain amyloid and tau pathologies.

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer's disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-ß peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-ß antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-ß antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer's pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-ß antibodies dramatically reduces amyloid-ß40 and amyloid-ß42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer's disease and other neurodegenerative disorders.

Reporter Immobilization Assay (REIA) for Bioconjugating Reactions.

Enzymes able to ligate biomolecules are emerging tools to generate site-specific bioconjugates. In this study we present a detection and screening method for bioconjugating enzymes which overcomes limitations of analytical methods such as HPLC or MS. These techniques are experimentally demanding and often limited in sensitivity and throughput compared to enzymatic assays. The principle of this Reporter Immobilization Assay (REIA) is the ligation of a reporter enzyme to a peptide carrying an affinity handle, which can be utilized for its isolation. The REIA system exhibits a high sensitivity with a linear range down to 1 µg/mL (55 nM), a variation coefficient of 6.5%, and can be performed cost-efficiently in 96-well microtiter plate format. The application of this assay allowed the characterization of a thiol transpeptidase sortase from S. aureus which is an important drug target and a biotechnological tool for ligation and modification of proteins. Thereby, yet-undetectable promiscuous activity of sortase could be detected, e.g., the acceptance of alanine as nucleophile. In addition, we were able to provide evidence that the REIA is suitable for high throughput screening of enzyme libraries using crude cellular extract with a throughput of 600 samples per hour.

Targeted delivery of interferon-α to hepatitis B virus-infected cells using T-cell receptor-like antibodies.

UNLABELLED: During antiviral therapy, specific delivery of interferon-α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T-cell receptor-like antibodies (TCR-L) able to selectively bind hepatitis B virus (HBV)-infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18-27) and surface (HBs183-91) HBV epitopes associated with different human leukocyte antigen (HLA)-A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR-L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV-peptide HLA-complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR-like variable regions of the antibodies. CONCLUSION: TCR-L antibodies can be used to target cytokines to HBV-infected hepatocytes in vitro. Fusion of IFNα to TCR-L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV-infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα.

Closing two doors of viral entry: intramolecular combination of a coreceptor- and fusion inhibitor of HIV-1.

We describe a novel strategy in which two inhibitors of HIV viral entry were incorporated into a single molecule. This bifunctional fusion inhibitor consists of an antibody blocking the binding of HIV to its co-receptor CCR5, and a covalently linked peptide which blocks envelope mediated virus-cell fusion. This novel bifunctional molecule is highly active on CCR5- and X4-tropic viruses in a single cycle assay and a reporter cell line with IC50 values of 0.03-0.05 nM. We demonstrated that both inhibitors contribute to the antiviral activity. In the natural host peripheral blood mononuclear cells (PBMC) the inhibition of CXCR4-tropic viruses is dependant on the co-expression of CCR5 and CXCR4 receptors. This bifunctional inhibitor may offer potential for improved pharmacokinetic parameters for a fusion inhibitor in humans and the combination of two active antiviral agents in one molecule may provide better durability in controlling the emergence of resistant viruses.

Structural basis of the adaptive molecular recognition by MMP9.

Matrix metalloproteinase (MMPs) are critical for the degradation of extracellular matrix components and, therefore, need to be regulated tightly. Almost all MMPs share a homologous C-terminal haemopexin-like domain (PEX). Besides its role in macromolecular substrate processing, the PEX domains appear to play a major role in regulating MMP activation, localisation and inhibition. One intriguing property of MMP9 is its competence to bind different proteins, involved in these regulatory processes, with high affinity at an overlapping recognition site on its PEX domain. With the crystal structure of the PEX9 dimer, we present the first example of how PEX domains accomplish these diverse roles. Blade IV of PEX9 mediates the non-covalent and predominantly hydrophobic dimerisation contact. Large shifts of blade III and, in particular, blade IV, accompany the dimerisation, resulting in a remarkably asymmetric homodimeric structure. The asymmetry provides a novel mechanism of adaptive protein recognition, where different proteins (PEX9, PEX1, and TIMP1) can bind with high affinity to PEX9 at an overlapping site. Finally, the structure illustrates how the dimerisation generates new properties on both a physico-chemical and functional level.

Crystal structures of uninhibited factor VIIa link its cofactor and substrate-assisted activation to specific interactions.

Factor VIIa initiates the extrinsic coagulation cascade; this event requires a delicately balanced regulation that is implemented on different levels, including a sophisticated multi-step activation mechanism of factor VII. Its central role in hemostasis and thrombosis makes factor VIIa a key target of pharmaceutical research. We succeeded, for the first time, in recombinantly producing N-terminally truncated factor VII (rf7) in an Escherichia coli expression system by employing an oxidative, in vitro, folding protocol, which depends critically on the presence of ethylene glycol. Activated recombinant factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of an inhibitor-free and sulphate-free, but isomorphous crystal form identified structural details of factor VIIa stimulation. The stabilisation of Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a sulphate ion appear to be sufficient to mimic the disorder-order transition conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped activity modulation dependent on ethylene glycol. The ethylene glycol-binding site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol binding induces a significant conformational rearrangement of the 60 loop. This region serves as a recognition site of the physiologic substrate, factor X, which is common to both factor VIIa and factor IXa. These results provide a mechanistic framework of substrate-assisted catalysis of both factor VIIa and factor IXa.

The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved.

We examined the influence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of differences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the effects on k(cat) depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not affect the S2-S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1-P1 interaction over substrate binding in the S2-S4 sites.

Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation.

The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.

Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.

CD4-BFFI: a novel, bifunctional HIV-1 entry inhibitor with high and broad antiviral potency.

Resistance to antiretroviral drugs is a common problem in the treatment of HIV-1-infected patients. To overcome resistance, we generated a novel, bifunctional HIV-1 entry inhibitor by combining the anti-CD4 monoclonal antibody (mAb) 6314 with a fusion inhibitor similar to T-651 (anti-CD4 mAb based BiFunctional Fusion Inhibitor, CD4-BFFI). CD4-BFFI has potent antiviral activity against a multitude of HIV-1 isolates independent of their co-receptor usage and genetic background. It has higher antiviral potency compared to the fusion inhibitor T-651 or the anti-CD4 mAb 6314 used independently. More importantly, every HIV-1 strain tested was fully inhibited by CD4-BFFI while many strains were only partially inhibited by 6314. CD4-BFFI also retained antiviral potency against virus strains resistant to two fusion inhibitors, a CCR5 antagonist and an anti-CCR5 mAb. Pre-incubation of cells with a saturating concentration of anti-CD4 mAbs reduced the antiviral potency of CD4-BFFI, suggesting that binding of CD4-BFFI to the cell surface via its CD4 mAb portion is required for the antiviral potency of its fusion inhibitor moiety. Collectively, we present a novel HIV-1 inhibitor with a dual mode of action and excellent antiviral potency against wildtype and entry-inhibitor resistant virus strains suggesting that CD4-BFFI may have a high barrier to resistance.

CD4-anchoring HIV-1 fusion inhibitor with enhanced potency and in vivo stability.

In this study, we describe a novel CD4-targeting bifunctional human immunodeficiency virus (HIV-1) fusion inhibitor (CD4-BFFI) that blocks HIV-1 entry by inhibiting both HIV-1 attachment and fusion and is highly potent against both R5 and X4 HIV-1 viruses in various antiviral assays, including peripheral blood mononuclear cell (PBMC) infection assays. Previously, we have reported a CCR5 antibody-based bifunctional HIV-1 fusion inhibitor (BFFI) that was highly active in blocking R5 HIV-1 infection but was ineffective against X4 viruses infecting human PBMCs (Kopetzki, E., Jekle, A., Ji, C., Rao, E., Zhang, J., Fischer, S., Cammack, N., Sankuratri, S., and Heilek, G. (2008) Virology J. 5, 56-65). CD4-BFFI, which consists of two HIV-1 fusion inhibitor (FI) T-651 variant peptides recombinantly fused to the Fc end of a humanized anti-CD4 monoclonal antibody, has demonstrated more than 100-fold greater antiviral activity than T-651 variant or the parental CD4 monoclonal antibody. Mechanistic studies revealed that CD4-BFFI primarily blocks the HIV-1-cell fusion step through its FI peptide moieties. The enhanced antiviral activity of CD4-BFFI is most likely due to avid binding of the bivalent FI peptides as well as the increased local concentration of CD4-BFFI via attachment to the target cell surface receptor CD4. In vivo pharmacokinetic studies demonstrated that CD4-BFFI was stable in monkey blood, and a dose of 10 mg/kg maintained serum concentrations greater than 2,000-fold over the IC(90) value for 7 days postdosing. This novel bifunctional inhibitor with improved potency and favorable pharmacokinetic properties may offer a novel approach for HIV-1 therapy.

Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop.

Coagulation factor IXa (fIXa) plays a central role in the coagulation cascade. Enzymatically, fIXa is characterized by its very low amidolytic activity that is not improved in the presence of cofactor, factor VIIIa (fVIIIa), distinguishing fIXa from all other coagulation factors. Activation of the fIXa-fVIIIa complex requires its macromolecular substrate, factor X (fX). The 99-loop positioned near the active site partly accounts for the poor activity of fIXa because it adopts a conformation that interferes with canonical substrate binding in S2-S4. Here we show that residues Lys-98 and Tyr-99 are critically linked to the amidolytic properties of fIXa. Exchange of Tyr-99 with smaller residues resulted not only in an overall decreased activity but also in impaired binding in S1. Replacement of Lys-98 with smaller and uncharged residues increased activity. Simultaneous mutagenesis of Lys-98, Tyr-177, and Tyr-94 produced an enzyme with 7000-fold increased activity and altered specificity. This triple mutant probably mimics the conformational changes that are physiologically induced by cofactor and substrate binding. It therefore provides a cooperative two-step activation model for fIXa. Tyr-177 locks the 99-loop in an inactive conformation which, in the physiologic complex, is released by cofactor fVIIIa. FX is then able to rearrange the unlocked 99-loop and subsequently binds to the active site cleft.