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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.
Mol Immunol 1995 Nov
PMID:In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5. 855 43

The present study set out to investigate whether phage display could be used to improve the properties of a high-affinity human monoclonal antibody directed against the third hypervariable loop (V3 loop) of human immunodeficiency virus (HIV). The aim was to increase affinity through slowing the dissociation rate (off-rate constant of koff), whilst retaining the ability of this antibody to bind diverse V3 loop sequences. When reformatted as a scFv, the antibody fragment retained the properties of the parental IgG, including the ability to neutralise virus. Heavy and light chains were sequentially replaced with repertoires of variable domains from non-immunised human donors followed by selection on biotinylated synthetic peptide. All selected variants derived from the same germline as the parental antibody. Variants of the light chain provided little if any improvement, whereas two residue changes in VHCDR2 and one in VHFR3 resulted in a reduced koff from gp120 protein of the MN strain (MNgp120) and synthetic V3 loop peptides as measured by surface plasmon resonance using the BIAcore instrument (Pharmacia Biosensor). VHCDR3 was modified using synthetic oligonucleotides and several clones with reduced koff identified, a number of different substitutions occurring at a single residue position. The residues in the heavy chain identified as reducing koff were simultaneously randomised by site-directed mutagenesis, resulting in scFv variants with koff slowed up to sevenfold. Far from compromising recognition of variant loops, binding to these sequences was improved; the koff from synthetic peptides modelled on V3 loop variants being slowed to a degree similar to that observed with MNgp120. All four changes were located towards either extremes of CDRs 2 and 3, suggesting that the mechanism of improvement may be one of alternation of loop conformation. This work illustrates that phage display can be used to tailor the properties of a therapeutic monoclonal antibody in a predefined fashion.
J Mol Biol 1996 Feb 16
PMID:Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity. 860 15

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.
Plant Mol Biol 1996 Feb
PMID:The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. 862 9

Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use.
Mol Immunol 1996 Feb
PMID:A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL. 864 42

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.
 ...
PMID:High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies. 881 24

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.
Plant Mol Biol 1996 Dec
PMID:Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. 898 May 48

While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.
J Mol Biol 1997 Jan 17
PMID:A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding. 902 Sep 80

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115). We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.
J Mol Biol 1997 Apr 25
PMID:Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation. 914 45

Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.
J Mol Biol 1997 May 09
PMID:Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions. 917 Dec 84

We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.
Mol Immunol 1996 Dec
PMID:Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase. 917 90


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