Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

Background  

Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.     

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ?-propionolactone (?-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.     

Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.     

Lactobacilli-expressed single-chain variable fragment (scFv) specific for intercellular adhesion molecule 1 (ICAM-1) blocks cell-associated HIV-1 transmission across a cervical epithelial monolayer

The vaginal and cervical epithelia provide an initial barrier to sexually acquired HIV-1 infection in women. To study the interactions between HIV-1-infected cells or cell-free HIV-1 and the reproductive epithelium, the transmission of HIV-1 by infected cells or cell-free virus across human cervical epithelial cells was examined using a Transwell culture system. Cell-associated HIV-1 was transmitted more efficiently than cell-free virus, and monocyte-associated virus was transmitted most efficiently. Abs to ICAM-1 added to the apical side of the epithelium blocked cell-mediated transepithelial HIV-1 transmission in vitro. When used in a previously described model of vaginal HIV-1 transmission in human PBL-SCID mice, anti-murine ICAM-1 Abs (0.4 microg/10 microl) also blocked vaginal transmission of cell-associated HIV-1 in vivo. To evaluate a candidate delivery system for the use of this Ab as an anti-HIV-1 microbicide, anti-ICAM single-chain variable fragment Abs secreted by transformed lactobacilli were evaluated for their protective efficacy in the Transwell model. Like the intact Ab and Fab derived from it, the single-chain variable fragment at a concentration of 6.7 microg/100 microl was able to reduce HIV-1 transmission by 70 +/- 5%. These data support the potential efficacy of an anti-ICAM Ab delivered by lactobacilli for use as an anti-HIV-1 microbicide.     

Expression of functional single-chain variable domain fragment (scFv) antibody against Metolcarb in Pichia pastoris

A Pichia pastoris system was used to express a single-chain variable fragment (scFv) antibody targeted against Metolcarb. The specific scFv gene was amplified from the phage-display scFv library and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C-scFv, was linearized and transformed into P. pastoris strain X-33. A transformant named X-33-Pp-SMW-12-6, which showed strong expression of antibodies, was isolated, and the culture conditions, including methanol induction concentrations, inoculum densities, and pH, were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of the scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against Metolcarb are comparable to those of scFv antibodies produced in E. coli. Recoveries of Metolcarb demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of Metolcarb residue in environmental and agricultural samples using a competitive inhibition enzyme-linked immunosorbent assay. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against Metolcarb for downstream applications.     

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

Background  

The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.     

Expression of functional single-chain variable domain fragment antibody (scFv) against mycotoxin zearalenone in Pichia pastoris

A synthetic gene coding for single-chain variable domain fragment antibody against mycotoxin zearalenone (scFv-ZEN) has been designed, constructed and expressed in Pichia pastoris. The native scFv-ZEN sequence was optimized to Pichia preference codon usage. The expression level of codon-optimized scFv-ZEN was slightly higher than that of native scFv-ZEN, and its maximum yield reached 328 mg total protein/l in flask culture. The binding activities of two selected clones to ZEN using surface plasmon resonance analysis were comparable or better than that of monoclonal antibody. Our results demonstrate the potential of soluble scFv-ZEN for developing a rapid and affordable immunoassay for detection of ZEN in food and feedstuff.     

Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75?h, typically between 140 and 160?g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5?% of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0?% boundary for longer periods of time when compared to a traditional proportional–integral–derivative algorithm, which tends to destabilize with increasing cell density.     

Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity

We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.     

Possible relationship between poly(ADP-ribose)synthetase and formation of antibody against acetylcholine receptors in patients with myasthenia gravis

We examined the relationship of the serum levels of antibody against acetylcholine receptors to the serum levels of 13 enzymes, including various hydrolytic enzymes, poly(ADP-ribose)synthetase (Poly(ADP-ribose)Syn), and sialyltransferase (NANA-trans), in patients with myasthenia gravis. The patients were divided into two groups, depending on the presence or absence of thymoma. In spite of the absence of significant difference in the absolute levels of individual enzymatic activities between the two groups, the network relationships of such enzymes were quite different between the two groups. Of the 13 enzymes examined, only Poly(ADP-ribose)Syn showed a weak but significant correlation with the level of the antibody in the patients without thymoma. A multivariate study more clearly suggested the relationship between the antibody formation and Poly(ADP-ribose)Syn in the patients without thymoma. Such observations were not found in the patients with thymoma.     

Cloning of a single-chain variable fragment (scFv) switching active plasminogen activator inhibitor-1 to substrate

Increased levels of plasminogen activator inhibitor-1 (PAI-1) are a well-known risk for cardiovascular diseases. A significant number of investigations are aimed at lowering plasma levels of PAI-1 to enhance endogenous fibrinolysis. We have recently generated monoclonal antibodies that neutralize PAI-1 activity by switching the inhibitory conformation to a substrate conformation. However, intact murine antibodies have quite some disadvantages for therapeutic use in man. In the current study, we describe the construction of a smaller antibody fragment derived from a monoclonal antibody (MA-8H9D4) with PAI-1 neutralizing properties. The cDNAs encoding the variable domains of the heavy and light chain were amplified, linked and cloned into a phagemid vector. Resulting clones were expressed as a single-chain variable fragment (scFv, V_H-(Gly₄Ser)₃-V_L) on the surface of a phage and selected for binding to PAI-1. Subsequently, a positive phage was used for the production of soluble scFv-8H9D4. Following purification, the characteristics of the scFv-8H9D4 were compared to those of the original MA-8H9D4. The scFv inhibited PAI-1 activity to a similar extent as MA-8H9D4 and by a similar mechanism, i.e., induction of a conformational switch. Thus, this smaller antibody fragment, exhibiting the same properties as the parent molecule may constitute a useful starting point for the design of PAI-1 neutralizing therapeutics. © 1997 Elsevier Science B.V. All rights reserved.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.     

Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization

We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection.     

Production and characterization of a bacterial single-chain antibody fragment specific to B-cell-activating factor of the TNF family

An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a (Gly4Ser)3 linker, using an assembly polymerase chain reaction. The construct VH-linker-VL was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli BL21(DE3) as inclusion bodies. After extraction from the E. coli cells, the inclusion bodies were solubilized and denatured in the presence of 8M urea. The expressed scFv fusion proteins were purified by Ni(2+)-IDA His-bind resin and finally renatured by dialysis. The purity and activity of the purified scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and enzyme-linked immunosorbent assay. The result revealed that the ABL-1 scFv retains the specific binding activity to BAFF with an affinity constant of 0.9x10(-8)molL(-1).