Tailoring structure-function and targeting properties of ceramides by site-specific cationization

In the course of our studies on compartment-specific lipid-mediated cell regulation, we identified an intimate connection between ceramides (Cers) and the mitochondria-dependent death-signaling pathways. Here, we report on a new class of cationic Cer mimics, dubbed ceramidoids, designed to act as organelle-targeted sphingolipids (SPLs), based on conjugates of Cer and dihydroceramide (dhCer) with pyridinium salts (CCPS and dhCCPS, respectively). Ceramidoids having the pyridinium salt unit (PSU) placed internally (alpha and gamma- CCPS) or as a tether (omega-CCPS) in the N-acyl moiety were prepared by N-acylation of sphingoid bases with different omega-bromo acids or pyridine carboxylic acid chlorides following capping with respective pyridines or alkyl bromides. Consistent with their design, these analogs, showed a significantly improved solubility in water, well-resolved NMR spectra in D(2)O, broadly modified hydrophobicity, fast cellular uptake, and higher anticancer activities in cells in comparison to uncharged counterparts. Structure-activity relationship (SAR) studies in MCF-7 breast carcinoma cells revealed that the location of the PSU and its overall chain length affected markedly the cytotoxic effects of these ceramidoids. All omega-CCPSs were more potent (IC(50/48 h): 0.6-8.0 microM) than their alpha/gamma-CCPS (IC(50/48 h): 8-20 microM) or D-erythro-C6-Cer (IC(50/48 h): 15 microM) analogs. omega-DhCCPSs were also moderately potent (IC(50/48 h): 2.5-12.5 microM). Long-chain omega-dhCCPSs were rapidly and efficiently oxidized in cells to the corresponding omega-CCPSs, as established by LC-MS analysis. CCPS analogs also induced acute changes in the levels and composition of endogenous Cers (upregulation of C16-, C14-, and C18-Cers, and downregulation of C24:0- and C24:1-Cers). These novel ceramidoids illustrate the feasibility of compartment-targeted lipids, and they should be useful in cell-based studies as well as potential novel therapeutics.     

Antigen recognition and targeted delivery by the single-chain Fv

The single-chain Fv (sFv) has proven attractive for immunotargeting, both alone and as a targeting element within sFv fusion proteins. This chapter summarizes the features of sFv proteins that have sparked this interest, starting with the conservation of Fv architecture that makes general sFv design practical. The length and composition of linkers used to bridge V domains are discussed based on the sFv literature; special emphasis is given to the (Gly₄Ser)₃ 15-residue linker that has proven of broad utility for constructing Fv regions of antibodies and other members of the immunoglobulin superfamily. The refolding properties of sFv proteins are summarized and examples given from our laboratory. Spontaneous refolding from the fully reduced and denatured state, typified by 26-10 sFv, is contrasted with disulfide-restricted refolding, exemplified by MOPC 315 and R11D10 sFv proteins, which recover antigen binding only if their disulfides have been oxidized prior to removal of denaturant. The medical value of sFv proteins hinges on their reliability in antigen recognition and rapidity in targeted delivery. Detailed analysis of specificity and affinity of antigen binding by the 26-10 antidigoxin sFv has demonstrated very high fidelity to the binding properties of the parent 26-10 sFv. These results gave confidence to the pursuit of more complex biomedical applications of these proteins, which is indicated by our work with the R11D10 sFv for the imaging of myocardial infarctions. Diagnostic imaging and therapeutic immunotargeting by sFv present significant opportunities, particularly as a result of their pharmacokinetic properties. Intravenously administered sFv offers much faster clearance than conventional Fab fragments or intact immunoglobulin with minimal background binding.     

Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage

Bacterial L-ASNases (L-asparaginases) catalyse the conversion of L-asparagine into L-aspartate and ammonia, and are widely used for the treatment of ALL (acute lymphoblastic leukaemia). In the present paper, we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsin-resistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at Arg206-Ser207 of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to histidine followed by covalent coupling of mPEG-SNHS [methoxypoly(ethylene glycol) succinate N-hydroxysuccinimide ester] to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared with the wild-type enzyme.     

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.     

Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïe phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.     

Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C). A growing body of evidence suggests that antibodies to PrP(C) can antagonize deposition of PrP(Sc). However, host tolerance hampers the induction of immune responses to PrP(C), and cross-linking of PrP(C) by bivalent anti-PrP antibodies is neurotoxic. In order to obviate these problems, we explored the antiprion potential of recombinant single-chain antibody (scFv) fragments. scFv fragments derived from monoclonal anti-PrP antibody 6H4, flagged with c-myc and His6 tags, were correctly processed and secreted by mammalian RD-4 rhabdomyosarcoma cells. When cocultured with cells secreting anti-PrP scFv, chronically prion-infected neuroblastoma cells ceased to produce PrP(Sc), even if antibody-producing cells were physically separated from target cells in transwell cultures. Expression of scFv with irrelevant specificity, or of similarly tagged molecules, was not curative. Therefore, eukaryotically expressed scFv exerts a paracrine antiprion activity. The effector functions encoded by immunoglobulin constant domains are unnecessary for this effect. Because of their small size and their monovalent binding, scFv fragments may represent candidates for gene transfer-based immunotherapy of prion diseases.     

Refolding of single-chain Fv by use of an antigen-coupled column

A single-chain variable fragment (scFv) antibody against bisphenol A was refolded using an antigen (bisphenol A)-coupled column. The refolding efficiency was compared with that used in dialysis. The refolding efficiency of the antigen-coupled column was about 50–60%, which was much higher than with dialysis, due to a decrease in the concentration of the refolding molecules and to the suppression of the aggregate formation.     

Stability engineering of antibody single-chain Fv fragments

The application of single-chain Fv fragments (scFv) in medicine and biotechnology places great demands on their stability. Only recently has attention been given to the production of highly stable scFvs, and in a number of examples it was found that such fragments indeed perform better during practical applications. The structural parameters influencing scFv stability are now beginning to be elucidated. This review summarizes progress in rational and evolutionary engineering methods, the structural implications of these results, as well as some examples where stability engineering has been successfully applied.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

PEGylation of therapeutic proteins

Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.     

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.     

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies

The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced approximately 3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.     

Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins

A subset of lipophillic bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are thought to be biologically transformed into reactive intermediates forming covalently modified, "tissue-bound" bile acids that can exert several toxic effects. We have generated a single-chain Fv fragment (scFv) as a probe to monitor DCA residues anchored on proteins. DNA fragments encoding the variable heavy (V(H)) and light (V(L)) domains of a mouse antibody raised against a DCA hapten (Ab #88) were cloned by rapid amplification of cDNA 5'-ends. These sequences were combined via a common linker sequence coding (Gly(4)Ser)(3) to construct a single scFv gene with the gene segments in the following order: 5'-V(H)-linker-V(L)-3'. This construct was subcloned into an antibody-expression vector, pEXmide 5; soluble scFv protein was then expressed in the bacterial periplasm of the XLOLR Escherichia coli strain. In a competitive enzyme-linked immunosorbent assay using DCA-coated microtiter plates, the scFv provided a dose-response curve for free DCA ranging between 2 and 5000 pg/assay. The scFv reacts similarly with the l-lysine adduct of DCA (cross-reactivity, 72%), while bile acids having a modified DCA steroid skeleton were well-discriminated (cross-reactivity, <1%). This scFv could also monitor trace amounts of DCA residues anchored on a protein through DCA acyl adenylate reactions, the likely reactive intermediate. The present scFv may be a useful tool for trace characterization of tissue-bound bile acids; this usefulness may be significantly enhanced by fusion with signal-generating proteins, such as alkaline phosphatase or green fluorescent protein.     

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria.     

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production and characterization of a human single-chain Fv to collagenase IV

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Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (V_H) and light (V_L) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)₂ was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv′)₂ have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.     

Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).     

Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.     

Sequence analysis,expression, and paratope characterization of a single-chain Fv fragment for the eukaryote ribosomal P proteins

The variable genes of monoclonal antibody (mAb) B10, specific for the C-terminal region of the eukaryotic ribosomal P protein, have been cloned as a single-chain Fv fragment (scFv) and expressed in Escherichia coli. The primary sequence of the variable regions of the B10 antibody, together with a detailed characterization of the reactive residues of the antigen, allowed the construction of a model of the paratope-epitope interaction, giving a first insight into the binding mechanisms of anti-P autoantibodies to their target peptides. The mAb and scFv could be useful for extensive P protein detection since both recognize the highly conserved motif DDxGF.