Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleases (RNases) are being employed as alternative cytotoxic proteins to the conventionally used ones such as ricin and Pseudomonas exotoxin. Mammalian RNases are attractive enzymes because of their comparable cytotoxicity when suitably directed and the likelihood of lower immunogenicity compared to plant and bacterial toxins. Bovine seminal RNase (BSRNase) is a member of the RNase superfamily, but differs in many interesting ways. Unlike the rest of the family it is dimeric, and possesses antitumor and immunosuppressive properties. These features make it a choice candidate for a single-chain antibody (scFv) based immunotoxin. This work describes preliminary data on the construction, expression in Escherichia coli and characterization of a tumor-specific scFv (directed against human placental alkaline phosphatase)-BSRNase chimeric molecule. It is shown that the created molecule has RNA degrading activity and antigen-binding activity when refolded from bacterial inclusion bodies.
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PMID:Targeting phosphodiesterases as a strategy for killing tumor cells. 773 30

This is the first study in which the complex of a monoclonal autoantibody fragment and its target, stem loop II of U1 snRNA, was investigated with enzymatic and chemical probing. A phage display antibody library derived from bone marrow cells of an SLE patient was used for selection of scFvs specific for stem loop II. The scFv specificity was tested by RNA immunoprecipitation and nitrocellulose filter binding competition experiments. Immunofluorescence data and immunoprecipitation of U1 snRNPs containing U1A protein, pointed to an scFv binding site different from the U1A binding site. The scFv binding site on stem loop II was determined by footprinting experiments using RNase A, RNase V1, and hydroxyl radicals. The results show that the binding site covers three sequence elements on the RNA, one on the 5' strand of the stem and two on the 3' strand. Hypersensitivity of three loop nucleotides suggests a conformational change of the RNA upon antibody binding. A three-dimensional representation of stem loop II reveals a juxtapositioning of the three protected regions on one side of the helix, spanning approximately one helical turn. The location of the scFv binding site on stem loop II is in full agreement with the finding that both the U1A protein and the scFv are able to bind stem loop II simultaneously. As a consequence, this recombinant monoclonal anti-U1 snRNA scFv might be very useful in studies on U1 snRNPs and its involvement in cellular processes like splicing.
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PMID:Characterization of an anti-RNA recombinant autoantibody fragment (scFv) isolated from a phage display library and detailed analysis of its binding site on U1 snRNA. 974 Jan 30

Immunotoxins consist of a target-cell-specific binding moiety, chemically or recombinantly linked to a cytotoxic component. A number of different immunotoxins (IT) have increasingly been evaluated for immunotherapy. Since these foreign proteins are highly immunogenic in human, we have developed recombinant IT using the human ribonuclease angiogenin. Due to their potential toxic effects on eucaryotic cells, these IT are usually expressed in bacteria. Depending on the structure, size, and sequence of the desired IT, bacterial expression can be limited and the yield after purification be unsatisfactory. Therefore, the expression of IT in eucaryotic cells could provide a promising alternative. For this purpose we genetically fused the anti-CD30 single-chain variable fragment (scFv) Ki4 to the N- and C-termini of recombinant angiogenin. Both IT possess leader sequences, which mediate their secretion into the cell culture supernatant. Using a bicistronic mRNA the IT were simultaneously expressed together with enhanced green fluorescent protein (EGFP). This allows direct monitoring of transfected cells. An additional plasmid encoded Zeocin resistance enhances the cultivation of transfected cells under selection pressure. Three days after transfection of 293T-cells, unpurified IT were analyzed by flow cytometry and competitive cell proliferation assays. This is the first report on the use of eucaryotic cells for the secretion of functionally active IT with a human effector domain.
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PMID:Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. 1269 83

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.
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PMID:Design of multivalent complexes using the barnase*barstar module. 1463 68

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (K(d) = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.
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PMID:A fully human antitumor immunoRNase selective for ErbB-2-positive carcinomas. 1525 57

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22(+) tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V(L)36(Leu-->Tyr)) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K(D)=0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22(+) tumor cell lines with high efficacy (IC(50)=3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.
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PMID:Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein. 1585 Aug 2

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.
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PMID:A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells. 1630 Sep 8

Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.
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PMID:Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. 1719 96

Human anti-ErbB2 immunoRNase with Erbicin fused to HP-RNase (ERB-hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti-ErbB2 scFv. It binds selectively with high affinity to ErbB2-positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB-hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB-hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.
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PMID:Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent. 1720 33

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.
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PMID:Anti-idiotypic antibody mimics proteolytic function of parent antigen. 1802 Apr 54


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