Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge.

UNLABELLED: Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE: Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect in vivo even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.

Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo.

The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

The activated form of gelatinase B/matrix metalloproteinase-9 is associated with diabetic vitreous hemorrhage.

We aimed to investigate the presence and activation status of matrix metalloproteinase (MMP)-2 and MMP-9 in vitreous samples from proliferative diabetic retinopathy (PDR) patients. Paired vitreous and serum samples were obtained from patients undergoing vitrectomy for the treatment of rhegmatogenous retinal detachment (RD) (65 patients) and PDR (67 patients). PDR patients were diagnosed for the presence of hemorrhage and/or patent new vessels. Quantitative assays were performed for vitreous protein content, MMP-2, MMP-9, tissue inhibitor of metalloproteinases-1 (TIMP-1) and hemoglobin. Qualitative evaluation of the MMP-2 and MMP-9 activation status was performed by zymography. Vitreous samples contained proMMP-2 but levels were unselectively related to total protein content. ProMMP-9 and activated MMP-9 levels were significantly increased in PDR patients (p<0.001 for both comparisons). In addition, TIMP-1 levels were significantly increased in PDR patients (p=0.004) and functionally inhibited activation of MMP-9 in vitreous samples. None of the parameters significantly differed between PDR patients with patent new vessels and those with inactive disease. However, activated MMP-9 levels in vitreous samples of PDR patients with hemorrhage (75.7+/-106.3 scanning units per 2 microl) were significantly higher than those in PDR patients without hemorrhage (7.1+/-16.2 scanning units per 2 microl) (p<0.001) and strongly correlated with hemoglobin levels (r=0.7525; p<0.001). Activated MMP-9 was not detected in paired serum samples. We conclude that activated MMP-9 might be involved in hemorrhagic transformation in patients with PDR.

Remnant epitopes, autoimmunity and glycosylation.

The role of extracellular proteolysis in innate and adaptive immunity and the interplay between cytokines, chemokines and proteinases are gradually becoming recognized as critical factors in autoimmune processes. Many of the involved proteinases, including those of the plasminogen activator and matrix metalloproteinase cascades, and also several cytokines and chemokines, are glycoproteins. The stability, interactions with inhibitors or receptors, and activities of these molecules are fine-controlled by glycosylation. We studied gelatinase B or matrix metalloproteinase-9 (MMP-9) as a glycosylated enzyme involved in autoimmunity. In the joints of rheumatoid arthritis patients, CXC chemokines, such as interleukin-8/CXCL8, recruit and activate neutrophils to secrete prestored neutrophil collagenase/MMP-8 and gelatinase B/MMP-9. Gelatinase B potentiates interleukin-8 at least tenfold and thus enhances neutrophil and lymphocyte influxes to the joints. When cartilage collagen type II is cleaved at a unique site by one of several collagenases (MMP-1, MMP-8 or MMP-13), it becomes a substrate of gelatinase B. Human gelatinase B cleaves the resulting two large collagen fragments into at least 33 peptides of which two have been shown to be immunodominant, i.e., to elicit activation and proliferation of autoimmune T cells. One of these two remnant epitopes contains a glycan which is important for its immunoreactivity. In addition to the role of gelatinase B as a regulator in adaptive immune processes, we have also demonstrated that it destroys interferon-beta, a typical innate immunity effector molecule and therapeutic cytokine in multiple sclerosis. Furthermore, glycosylated interferon-beta, expressed in Chinese hamster ovary cells, was more resistant to this proteolysis than recombinant interferon-beta from bacteria. These data not only prove that glycosylation of proteins is mechanistically important in the pathogenesis of autoimmune diseases, but also show that targeting of glycosylated proteinases or the use of glycosylated cytokines seems also critical for the treatment of autoimmune diseases.

Gelatinase B/matrix metalloproteinase-9 provokes cataract by cleaving lens betaB1 crystallin.

Cataract is a common cause of blindness and results from destruction of the microarchitecture of the lens. It is observed in many genetic syndromes, infections, inflammatory diseases and during aging. Fluctuations in lens density and light scattering by altered refraction index form the physical basis for this process, but the pathogenesis is poorly understood. Increased levels of gelatinase B/matrix metalloproteinase-9 have been reported for cataract-associated disorders such as eye inflammation and diabetes. We demonstrate that incubation of lenses with gelatinase B leads immediately to cataract. In complete eye extracts, betaB1 crystallin was identified as the major gelatinase B substrate by combination of proteomics, mass spectrometry, and Edman degradation analysis. The cleavage of betaB1 crystallin was also observed in vivo after endogenous gelatinase B-induction by the chemokine granulocyte chemotactic protein-2 in wild-type mice but not in gelatinase B-/- mice.

In vivo activation of gelatinase B/MMP-9 by trypsin in acute pancreatitis is a permissive factor in streptozotocin-induced diabetes.

Matrix metalloproteinases, in particular gelatinase B/MMP-9, are key mediators in autoimmune diseases like multiple sclerosis and rheumatoid arthritis, but their pathogenic roles in diabetes are not well established. Gelatinase B has previously been shown to be upregulated in pancreas tissue from patients with acute and chronic pancreatitis and was suggested to exacerbate diabetes by cleaving insulin. In this study, the role of gelatinase B in diabetes was investigated using two streptozotocin-induced animal models of type I diabetes. In both a hyperacute and a subacute model, gelatinase B upregulation was found to be associated with disease activity. However, gelatinase B deficiency did not significantly protect against diabetes development, and wild-type and gelatinase B-deficient animals behaved similarly in terms of beta-cell apoptosis or necrosis. The fact that gelatinase B was found almost exclusively as the inactive pro-enzyme in most of the streptozotocin-induced diabetic animals may explain the lack of a gelatinase B effect. On the contrary, gelatinase B was completely activated in a minority (15%) of wild-type animals. This coincided with exocrine pancreatic inflammation, as revealed by the presence of active trypsin. The discovery of in vivo activation of progelatinase B by trypsin in acute pancreatitis is extended in a model of caerulein-induced pancreatitis. In the latter model, trypsinogen activation is systematically achieved and gelatinase B is found in its active form. In conclusion, gelatinase B itself is not a causative factor but, when activated by endogenous trypsin, is a permissive factor for insulin degradation and diabetes.

Chemokines and gelatinases in the aqueous humor of patients with active uveitis.

PURPOSE: To investigate the involvement of the chemokines CXCL10/IP-10, CXCL11/I-TAC, CXCL8/1L-8, CXCL6/GCP-2, CCL3/MIP-1alpha, and CCL18/PARC, and gelatinases A and B in uveitis. DESIGN: Prospective, experimental, case-control study. METHODS: Aqueous humor samples from 30 patients with active uveitis, and 14 control patients and paired serum samples were assayed for chemokines with specific enzyme-linked immunosorbent assays (ELISAs) and for gelatinase levels by quantitative zymography. RESULTS: In control AH, none of the chemokines was detected. Gelatinase A was detected in all samples, and gelatinase B was detected in only one sample. In patients with uveitis, IP-10 was detected in all AH samples, whereas I-TAC, IL-8, GCP-2, MIP-1alpha, and PARC were detected in three, 16, six, two, and 12 samples, respectively. IP-10 levels were significantly higher in AH samples than those of serum (P =.006). Gelatinase A was detected in 29 AH samples and gelatinase B was detected in 26 samples. Gelatinase A levels were significantly higher in AH samples from patients than those of controls (P <.0001). In 11 AH samples, gelatinase B was detected in complex with lipocalin (NGAL). Disease activity correlated significantly with the levels of IP-10 (r =.627; P <.0001), gelatinase A (r =.508; P =.002), gelatinase B (r =.685; P <.0001), and NGAL-gelatinase B complex (r =.595; P <.0001). CONCLUSIONS: These data suggest a pathogenic role of the T lymphocyte chemoattractant IP-10 and gelatinases in the recruitment and activity of T cells into the eye in patients with uveitis and in the pathogenesis of uveitis.

Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin.

Genetic, endocrine, and environmental factors contribute to the development of diabetes. Much information has been gathered on the homeostasis mechanisms of glucose regulation by insulin-producing pancreatic beta cells. Here we demonstrate high expression levels of gelatinase B (matrix metalloproteinase-9, MMP-9) by neutrophils in acute pancreatitis and by ductular epithelial cells in chronic pancreatitis. Because gelatinase B processes cytokines and chemokines, we investigated whether and how gelatinase B cleaves insulin. Pure human neutrophil gelatinase B was found to destroy insulin by cleavage at 10 sites. Pancreatic islet and ductular cells are relatively spared in comparison with the complete destruction of acinar cells of the exocrine pancreas in chronic pancreatitis. High expression levels of gelatinase B are maintained in the immediate proximity of insulin-secreting beta cells. Consequently, diabetes may be worsened by enzymatic degradation of insulin by gelatinase B and by the consequent enhancement of the autoimmune process. Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin.

Remnant epitopes generate autoimmunity: from rheumatoid arthritis and multiple sclerosis to diabetes.

Autoimmune diseases are characterized by inflammation and by the development and maintenance of antibodies and T lymphocytes against "self" antigens. Although the etiology of these diseases is unknown, they have a number of cellular and molecular mechanisms in common. Pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are upregulated and activate the inflammatory process. Chemokines recruit and activate leukocytes to release proteases, including matrix metalloproteinases (MMPs). These proteases degrade proteins into remnant fragments, which often constitute immunodominant epitopes. Either by direct loading into major histocompatibility complex (MHC) molecules or after classical antigen uptake, processing and MHC presentation, these remnant epitopes are presented to autoreactive T lymphocytes. Also, posttranslationally modified remnant peptides may stimulate B cells to produce autoantibodies. This forms the basis of the "Remnant Epitopes Generate Autoimmunity" (REGA) model. We have documented evidences for this model in multiple sclerosis (MS), rheumatoid arthritis (RA) and diabetes, which are summarized here. Furthermore, three topics will be addressed to illustrate the importance of glycobiology in the pathogenesis of autoimmune diseases. In MS, gelatinase B or MMP-9 is a pathogenic glycoprotein of which the sugars contribute to its interactions with the tissue inhibitor of metalloproteinases-1 (TIMP-1) and thus assist in the determination of the enzyme activity. In RA, gelatinase B cleaves denatured type II collagen into remnant epitopes, some of which constitute immunodominant glycopeptides. This implies that immunodominant epitope scanning experiments should preferably be done with natural posttranslationally modified glycopeptides, rather than with unmodified (synthetic) peptides. Sugars can also be used as molecular probes to induce autoimmune diseases. One of the best examples is the induction of acute pancreatitis, insulitis and diabetes by streptozotocin. In addition, gelatinase B is upregulated in pancreatitis and cleaves insulin. The most efficient cleavage by gelatinase B leads to a major insulin remnant epitope.