scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.
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Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent. (+info)
The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli.
(26/1180)
In a systematic study of the periplasmic folding of antibody fragments in Escherichia coli, we have analysed the expression of an aggregation-prone and previously non-functional anti-phosphorylcholine antibody, T15, as a model system and converted it to a functional molecule. Introduction of heavy chain framework mutations previously found to improve the folding of a related antibody led to improved folding of T15 fragments and improved physiology of the host E.coli cells. Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved folding and bacterial host physiology, but no improvement was seen in the wild type, suggesting the existence of a hierarchy in sequence positions leading to aggregation. Rational mutagenesis of the T15 light chain led to the production of functional T15 fragments for the first time, with increased levels of functional protein produced from V(H) manipulated constructs. We propose that a hierarchical analysis of the primary amino acid sequence, as we have described, provides guidelines on how correctly folding, functional antibodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation. (+info)
Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.
(27/1180)
Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens. (+info)
Methods to avoid adverse effect of circulating antigen on biodistribution of 125I-labeled antiTac dsFv: preinjection of intact antibody versus clearance of antigen with adivin-biotin system.
(28/1180)
The presence of circulating antigen may adversely affect the biodistribution of a radiolabeled antibody. The alpha subunit of the interleukin-2 receptor (IL-2Ralpha) is a cell-surface receptor that is overexpressed in various hematologic malignancies and in benign disorders. This receptor is cleaved from the cell surface and can be found in high concentrations in serum. Radiolabeled antiTac antibodies are being evaluated to target this receptor. Previous studies have shown that circulating soluble IL-2Ralpha (slL-2Ralpha) adversely affected the biodistribution of radiolabeled antiTac disulfide-stabilized (ds)Fv. In this study, we compared blocking and clearing sIL-2Ralpha to see which better minimized its interference with the biodistribution of radiolabeled antiTac dsFv. METHODS: Two models of sIL-2Ralpha were used: one consisted of mice given intravenous sIL-2Ralpha and the other consisted of mice bearing SP2/Tac tumor xenografts (IL-2Ralpha positive), which shed sIL-2Ralpha. We biotinylated humanized antiTac monoclonal antibody (bt-HuTac) and radiolabeled it with 125I. We then compared its biodistribution with that of humanized antiTac monoclonal antibody IgG (HuTac). We examined the biodistribution of an injected dose of 125I-labeled antiTac dsFv after a preinjection of HuTac to block the sIL-2Ralpha epitope and after a preinjection of bt-HuTac, followed by an avidin chase. RESULT: The 125I-labeled bt-HuTac cleared from the serum at a rate similar to that of HuTac. The avidin chase effectively cleared >92% of circulating 125I-labeled bt-HuTac within 20 min and was also effective in clearing sIL-2Ralpha. In comparison, HuTac prolonged the retention of 125I-labeled sIL-2Ralpha in the circulation, and the avidin chase decreased 125I-labeled sIL-2Ralpha to <18% of control. Although the two-step antigen-clearing system effectively cleared the antigen from the circulation and improved the biodistribution of 125I-labeled dsFv, the HuTac preinjection method had a similar but longer lasting beneficial effect on 125I-labeled dsFv biodistribution. CONCLUSION: Preinjection of either HuTac or bt-HuTac with avidin chase improved the biodistribution of subsequently administered 125I-labeled antiTac dsFv by preventing the dsFv from binding to the sIL-2Ralpha, but the HuTac blocking method is simpler and longer lasting. (+info)
Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection.
(29/1180)
Human immunodeficiency virus type 1 (HIV-1) encodes several proteins that are packaged into virus particles. Integrase (IN) is an essential retroviral enzyme, which has been a target for developing agents to inhibit virus replication. In previous studies, we showed that intracellular expression of single-chain variable antibody fragments (SFvs) that bind IN, delivered via retroviral expression vectors, provided resistance to productive HIV-1 infection in T-lymphocytic cells. In the current studies, we evaluated simian-virus 40 (SV40) as a delivery vehicle for anti-IN therapy of HIV-1 infection. Prior work suggested that delivery using SV40 might provide a high enough level of transduction that selection of transduced cells might be unnecessary. In these studies, an SV40 expression vector was developed to deliver SFv-IN (SV(Aw)). Expression of the SFv-IN was confirmed by Western blotting and immunofluorescence staining, which showed that > 90% of SupT1 T-lymphocytic cells treated with SV(Aw) expressed the SFv-IN protein without selection. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression and syncytium formation, was potently inhibited in cells expressing SV40-delivered SFv-IN. Levels of inhibition of HIV-1 infection achieved using this approach were comparable to those achieved using murine leukemia virus (MLV) as a transduction vector, the major difference being that transduction using SV40 did not require selection in culture whereas transduction with MLV did require selection. Therefore, the SV40 vector as gene delivery system represents a novel therapeutic strategy for gene therapy to target HIV-1 proteins and interfere with HIV-1 replication. (+info)
Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves.
(30/1180)
To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84. 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA. (+info)
Construction of a pseudoreceptor that mediates transduction by adenoviruses expressing a ligand in fiber or penton base.
(31/1180)
Modification of adenovirus to achieve tissue specific targeting for the delivery of therapeutic genes requires both the ablation of its native tropism and the introduction of specific, novel interactions. Inactivation of the native receptor interactions, however, would cripple the virus for growth in production cells. We have developed an alternative receptor, or pseudoreceptor, for the virus which might allow propagation of viruses with modified fiber proteins that no longer bind to the native adenovirus receptor (coxsackievirus/adenovirus receptor [CAR]). We have constructed a membrane-anchored single-chain antibody [m-scFv(HA)] which recognizes a linear peptide epitope (hemagglutinin [HA]). Incorporation of HA within the HI loop of the fiber protein enabled the modified virus to transduce pseudoreceptor expressing cells under conditions where fiber-CAR interaction was blocked or absent. The pseudoreceptor mediated virus transduction with an efficiency similar to that of CAR. In addition, the HA epitope mediated virus transduction through interaction with the m-scFv(HA) when it was introduced into penton base. These findings indicate that cells expressing the pseudoreceptor should support production of HA-tagged adenoviruses independent of retaining the fiber-CAR interaction. Moreover, they demonstrate that high-affinity targeting ligands may function following insertion into either penton base or fiber. (+info)
Selection of antibodies for intracellular function using a two-hybrid in vivo system.
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Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences. (+info)