Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants.RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants and young children (28,31). RSV belongs to the genus Pneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae. It is an enveloped, nonsegmented negative-strand RNA virus encoding 11 proteins, including nucleocapsid proteins (N, P, and L), surface proteins (F and G), and a matrix protein (M). In addition, the genome encodes two nonstructural proteins (NS1 and NS2), the functions of which are less clearly defined. RSV primarily infects epithelial cells of the respiratory tract and replicates exclusively in the cytoplasm. Progeny RSV particles exit the host cell by budding through the apical surfaces of polarized cells (35).In order to combat such infections, the immune system has evolved a potent antiviral response. Mediators, known as the type I interferons (alpha interferon [IFN-␣] and IFN-␤), stimulate the production of a range of antiviral gene products that limit virus replication and spread (4, 22). The type I IFN receptor consists of two subunits, IFNAR1 and IFNAR2, which are associated with the Janus kinases JAK1 and TYK2, respectively (23). Activation of these receptor tyrosine kinases results in tyrosine phosphorylation of signal transducer and activator of transcription 2 (STAT2) and STAT1. Activated STAT2 and STAT1 associate with interferon regulatory factor 9 (IRF-9) to form the transcriptional activator complex interferon-stimulated gene factor 3 (ISGF-3). These complexes translocate to the nucleus and bind IFN-stimulated response elements (ISRE) to initiate gene transcription and therefore antiviral immunity (8).Wild-type RSV induces a weak type I IFN response following infection (27), suggesting that it has the capacity to evade this host defense mechanism in order to establish a successful infection. RSV is thought to block IFN-␣ and -␤ signaling...

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5-11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.The Siglec 2 family of receptors consists of sialoadhesin (Siglec 1/Sn), CD22 (Siglec 2), CD33 (Siglec 3), myelin-associated glycoprotein (MAG or Siglec 4), and Siglecs 5-11. CD33-related Siglecs (Siglecs 5-11) are characterized by an N-terminal V-set Ig domain that mediates sialic acid binding and varying numbers of C2-set Ig domains. Sialic acids are a family of 9-carbon sugars that are derivatives of neuraminic acid or ketodeoxynonulosonic acid. The CD33-related Siglecs contain an ITIM and an ITSM in their cytoplasmic tails (1). The ITIM consists of a tyrosine with a leucine or valine at the ϩ3 position and a hydrophobic base at the Ϫ2 position ((I/L/V)XYXX(L/ V)), where X denotes any amino acid. The ITIM tyrosine is phosphorylated by Src family protein tyrosine kinases followed by recruitment of SH2-containing phosphatases such as the inositol polyphosphate 5-phosphatase, SHIP, and protein tyrosine phosphatases SHP-1 and SHP-2 to inhibit signaling (2). Engagement of Siglec 7 exerts an inhibitory signal mediated by tyrosine phosphorylation of the ITIM and recruitment of SHP-1 resulting in regulation of NK-cell-mediated target cell lysis, reduced phosphorylation of ZAP-70, and transcriptional activity of NFAT (nuclear factor of activated T cells) (4 -6). Cross-linking Siglec 7 positive chronic myeloid leukemia cells leads to an inhibitory effect on cell proliferation, suggesting that Siglec 7 may regulate normal myelopoiesis and could be a potential therapeutic target for chronic myeloid leukemia cells (7).Suppressor of cytokine signaling (SOCS) molecules inhibit the JAK/STAT pathway, ensuring that cytokine responses are regulated. SOCS proteins (CIS and SOCS1-7) contain an SH2 domain and a SOCS box, which binds Elongin B/C and Cul5/ Rbx1/2 (8). This complex can act as an E3 ligase and degrade associated proteins via the...

Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Background: Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.

BackgroundProteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting.ResultsHere we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect.ConclusionsThis data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L -like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L -like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L -like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L -like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis. [Mol Cancer Ther 2008;7(3):538 -47]

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International audienceUse of paramagnetic particles to isolate molecules or cells from complex media is well established. Typically, particles are manufactured and coated with a biological molecule which confers specific biorecognition. Incubation of particles with sample and exposure to magnetic fields isolates the species of interest. We designed, produced and assessed magnetized fusion proteins consisting of the antigen binding portion of an antibody (single chain Fv; scFv) fused to the heavy chain of the iron binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. The fusion proteins were chemically magnetized by introducing a paramagnetic iron core. The resultant fusion protein was shown to be magnetizable as well as capable of binding target antigens. These “organic” magnetizable particles possess a number of theoretical advantages over traditional inorganic particles