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Query: UNIPROT:P06889 (Mol)
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Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.
Mol Immunol 1996 Dec
PMID:Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv. 917 98

The generation of new antibodies for diagnostic applications using phage display could greatly decrease the time and expense of new assay development but, to be effective, the process must yield antibodies with desired specificity and affinity comparable to those obtained by monoclonal antibody technology. In order to evaluate the ability of the phage selection process to yield antibodies with the desired specificity and affinity, a family of anti-phenobarbital antibodies were cloned as scFvs and Fabs and displayed on M13 gene III fusion proteins. All of the antibodies are derived from similar germline VL and VH genes and exhibit extensive sequence homology except in VH CDR3. Despite these similarities, the range of panning efficiencies was observed to vary by two orders of magnitude for expressed scFvs. Unexpectedly, the scFv with the highest panning efficiency has the lowest affinity. In competitive panning experiments this scFv is preferentially isolated over higher affinity antibodies. This scFv expresses high levels of soluble binding protein while higher affinity scFvs express lower levels of protein or nonfunctional protein. These results suggest that the efficiency of functional expression of scFv proteins can greatly influence the type of antibody selected by phage display. The range of panning efficiency for functional Fabs was significantly less (four-fold) than that observed for scFvs and did not correlate to the expression level of the secreted proteins. Based on the results of the Fabs examined, it is concluded that the expression properties of Fabs may not exhibit the extent of variability observed for scFvs when displayed and use of an Fab architecture may provide an advantage over scFv architecture in the selection process. The feasibility of selecting against undesirable cross-reactivities has also been demonstrated by simple modification of phage panning conditions. These combined results support the concept of obtaining antibodies with desirable specificity and affinity for diagnostic applications through the use of phage display.
J Mol Recognit
PMID:Analysis of antibody selection by phage display utilizing anti-phenobarbital antibodies. 917 66

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti-neuraminidase NC10 scFv-streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcore biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti-ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.
Biochem Mol Biol Int 1997 Sep
PMID:Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents. 930 36

Proteins and peptides can be displayed on bacterial and bacteriophage surfaces as fusions to bacterial integral membrane proteins or phage coat proteins. We now report on the expression of peptide antigens on the surface of F pili, elaborated by F+ strains of Escherichia coli. The peptides were expressed as fusions to F pilin, the building block of the F pilus that is encoded by the traA gene on the F plasmid. Filamentous bacteriophage infection of E. coli is normally mediated by phage binding to pilin at the F pili tip. Expression of 13 to 15 amino acid long peptides on the F pilus completely blocked infection by derivatives of wild-type infectious M13 phage. However, when a phage displaying a specific recombinant antibody fragment was allowed to interact with F pili displaying an antigenic peptide a bacterial infection could be demonstrated. This infection, mediated by the antibody-antigen interaction, resulted in bacterial cells containing plasmids encoding both the protein and the ligand. In a model library, where a scFv antibody against the human cytomegalovirus AD-2 epitope was selected we achieved an enrichment of 2500 of phage carrying the specific antibody, indicating an efficient selective infection.
J Mol Biol 1997 Oct 31
PMID:Selective phage infection mediated by epitope expression on F pilus. 935 45

The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available. In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters. A group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters. In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis. Levels of expression were dependent on the sequence of VL used. Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original VH and VL regions are required for hapten binding, although the VH is more crucial for catalysis. By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies.
Mol Immunol
PMID:Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies. 946 25

We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation. Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.
J Mol Biol 1998 Jan 16
PMID:Antibody scFv fragments without disulfide bonds made by molecular evolution. 946 7

We cloned complementary DNA (cDNA) encoding the variable regions of heavy chain (VH) and of light chain (VL) of a monoclonal anti-carcinoembryonic antigen (CEA) antibody cross-reactive with nonspecific cross-reacting antigen-95 (NCA-95), which had been previously prepared and designated as CEA 79 (gamma 2a, kappa). From these cDNAs, a phagemid expression vector for the CEA79 single chain variable fragment (scFv) was generated. Enzyme-linked immunosorbent assay (ELISA), competitive ELISAs, and Western blotting confirmed that the scFv displayed on the surface of the bacteriophage had retained affinity for CEA and NCA-95. We then determined the nucleotide sequences of the cloned cDNAs for VH and VL. The sequence analysis revealed that VH and VL of the CEA 79 antibody represent new members of the mouse heavy chain subgroup "miscellaneous" and the kappa light chain subgroup "V", respectively.
Mol Cells 1997 Dec 31
PMID:Cloning and characterization of cDNAs encoding VH and VL of a monoclonal anti-CEA antibody (CEA 79) cross-reactive with NCA-95 and generation of a single-chain Fv molecule (scFv). 950 26

The structure of the complex between a recombinant single-chain Fv construct of antibody NC10 with a five-residue peptide linker between VH and VL (termed scFv(5)), and its antigen, tetrameric neuraminidase from influenza virus (NA), has been determined and refined at 2.5 A resolution. The antibody-antigen binding interface is very similar to that of a similar NC10 scFv-NA complex in which the scFv has a 15-residue peptide linker (scFv(15)), and the NC10 Fab-NA complex. However, scFv(5) and scFv(15) have different stoichiometries in solution. While scFv(15) is predominantly monomeric in solution, scFv(5) forms dimers exclusively, because the five-residue linker is not long enough to permit VH and VL domains from the same polypeptide associating and forming an antigen-binding site. Upon forming a complex with NA, scFv(15) forms a approximately 300 kDa complex corresponding to one NA tetramer binding four scFv(15) monomers, while scFv(5) forms a approximately 590 kDa complex, corresponding to two NA tetramers crosslinked by four bivalent scFv(5) dimers. However, the dimeric scFv(5) in the scFv(5)-NA crystals does not crosslink NA tetramers, and modelling studies indicate that it is not possible to pack four dimeric and simultaneously bivalent scFvs between the NA tetramers with only a five-residue linker between VH and VL. The inability arises from the exacting requirement to orient the two antigen-binding surfaces to bind the tetrameric NA antigen while avoiding steric clashes with NC10 scFv(5) dimers bound to other sites on the NA tetramer. The utility of bivalent or bifunctional scFvs with short linkers may therefore be restricted by the steric constraints imposed by binding multivalent antigens.
J Mol Biol 1998 Jun 19
PMID:Three-dimensional structures of single-chain Fv-neuraminidase complexes. 964 70

Recombinant antibody fragments expressed in the cytoplasm of cells have considerable practical potential. However in the reducing environment of the cytoplasm, the intradomain disulphide bonds are not formed and the fragments are unstable and expressed in low yields. Here we attempted to overcome these limitations. We first isolated an antibody single chain Fv fragment that binds and activates an inactive mutant beta-galactosidase. We then subjected the gene encoding the scFv fragment to random mutation in vitro by error-prone polymerase chain reaction, and co-expressed the mutant beta-galactosidase and mutant antibody fragments in lac- bacteria. By plating on limiting lactose, we selected for antibody mutants with improved expression, and after four successive rounds of mutation and selection, isolated an antibody fragment that is expressed in the bacterial cytoplasm with yields of 0.5 g/l in a shaker flask (A600 nm of 5.5) and 3.1 g/l (A600 nm=33) in a fermentor. Analysis of the mutant antibody fragments revealed that the disulphide bonds are reduced in the cytoplasm, and that the fragments could be denatured and renatured efficiently under reducing conditions in vitro. This shows that with a suitable method of screening or selection, it is possible to make folded and functional antibody fragments in excellent yield in the cytoplasm.
J Mol Biol 1998 Jul 03
PMID:Expression of an antibody fragment at high levels in the bacterial cytoplasm. 965 35

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains of Staphylococcus aureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein delta pIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.
Mol Biotechnol 1998 Jun
PMID:Filamentous bacteriophage display of a bifunctional protein A::scFv fusion. 971 79


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