Integrative expression system for delivery of antibody fragments by lactobacilli.

A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.

A detailed mathematical model predicts that serial engagement of IgE-Fc epsilon RI complexes can enhance Syk activation in mast cells.

The term serial engagement was introduced to describe the ability of a single peptide, bound to a MHC molecule, to sequentially interact with TCRs within the contact region between a T cell and an APC. In addition to ligands on surfaces, soluble multivalent ligands can serially engage cell surface receptors with sites on the ligand, binding and dissociating from receptors many times before all ligand sites become free and the ligand leaves the surface. To evaluate the role of serial engagement in Syk activation, we use a detailed mathematical model of the initial signaling cascade that is triggered when FcepsilonRI is aggregated on mast cells by multivalent Ags. Although serial engagement is not required for mast cell signaling, it can influence the recruitment of Syk to the receptor and subsequent Syk phosphorylation. Simulating the response of mast cells to ligands that serially engage receptors at different rates shows that increasing the rate of serial engagement by increasing the rate of dissociation of the ligand-receptor bond decreases Syk phosphorylation. Increasing serial engagement by increasing the rate at which receptors are cross-linked (for example by increasing the forward rate constant for cross-linking or increasing the valence of the ligand) increases Syk phosphorylation. When serial engagement enhances Syk phosphorylation, it does so by partially reversing the effects of kinetic proofreading. Serial engagement rapidly returns receptors that have dissociated from aggregates to new aggregates before the receptors have fully returned to their basal state.

Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior after experimental ischemic stroke. METHODS: Focal cerebral ischemia was produced by occluding the middle cerebral artery using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 hours. At 2(1/4) hours and 3 hours after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg/kg) or saline was injected through the tail vein. Sensorimotor function and infarction volume were assessed at 24 hours after ischemia. RESULTS: Administration of Fv-Hsp70 after focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensorimotor function compared with the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. CONCLUSIONS: Fv-Hsp70 protected the ischemic brain in this experimental stroke model.

A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.

Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.

Transgenic expression of single-chain anti-CTLA-4 Fv on beta cells protects nonobese diabetic mice from autoimmune diabetes.

T cell-mediated immunodestruction of pancreatic beta cells is the key process responsible for both the development of autoimmune diabetes and the induction of rejection during islet transplantation. In this study, we investigate the hypothesis that transgenic expression of an agonistic, membrane-bound single-chain anti-CTLA-4 Fv (anti-CTLA-4 scFv) on pancreatic beta cells can inhibit autoimmune processes by selectively targeting CTLA-4 on pathogenic T cells. Strikingly, transgenic expression of anti-CTLA-4 scFv on pancreatic beta cells significantly protected NOD mice from spontaneous autoimmune diabetes. Interestingly, local expression of this CTLA-4 agonist did not alter the diabetogenic properties of systemic lymphocytes, because splenocytes from transgenic mice or their nontransgenic littermates equally transferred diabetes in NOD/SCID recipients. By analyzing the T cell development in anti-CTLA-4 scFv/Th1/Th2 triple transgenic mice, we found that beta cell-specific expression of CTLA-4 agonist did not affect the development of Th1/Th2 or CD4(+)CD25(+) regulatory T cells. Most strikingly, islets from transgenic mice inhibited T cell response to immobilized anti-CD3 in a T cell-islet coculture system, suggesting a trans-mediated inhibition provided by transgenic islets. Finally, transgenic islets implanted in diabetic recipients survived much longer than did wild-type islets, indicating a therapeutic potential of this genetically modified islet graft in autoimmune diabetes.

T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.

BACKGROUND & AIMS: The final goal in hepatitis B therapy is eradication of the hepatitis B virus (HBV) replication template, the so-called covalently closed circular DNA (cccDNA). Current antiviral treatment of chronic hepatitis B depends on interferon alpha or nucleoside analogues inhibiting the viral reverse transcriptase. Despite treatment, cccDNA mostly persists in the host cell nucleus, continues to produce hepatitis B surface antigen (HBsAg), and causes relapsing disease. We therefore aimed at eliminating persistently infected hepatocytes carrying HBV cccDNA by redirecting cytolytic T cells toward HBsAg-producing cells. METHODS: We designed chimeric T-cell receptors directed against HBV surface proteins present on HBV-infected cells and used them to graft primary human T cells with antibody-like specificity. The receptors were composed of a single chain antibody fragment directed against HBV S or L protein fused to intracellular signalling domains of CD3xi and the costimulatory CD28 molecule. RESULTS: Our results show that these chimeric receptors, when retrovirally delivered and expressed on the cell surface, enable primary human T cells to recognize HBsAg-positive hepatocytes, release interferon gamma and interleukin 2, and, most importantly, lyse HBV replicating cells. When coincubated with HBV-infected primary human hepatocytes, these engineered, antigen-specific T cells selectively eliminated HBV-infected and thus cccDNA-positive target cells. CONCLUSIONS: Elimination of HBV cccDNA-positive hepatocytes following antiviral therapy is a major therapeutic goal in chronic hepatitis B, and adoptive transfer of grafted T cells provides a promising novel therapeutic approach. However, T-cell therapy may also cause liver damage and therefore needs further preclinical evaluation.

Functional mapping and single chain construction of the anti-cytokeratin 8 monoclonal antibody TS1.

The anti-cytokeratin (CK) 8 monoclonal antibody (mab) TS1 has been shown to efficiently bind to CK8 expressed in carcinomas in vivo. The anti-idiotypic antibody of TS1, alphaTS1, can be used to regulate the tumor:non-tumor ratio of TS1 by clearing non-tumor binding TS1 from the circulation. If the interaction of TS1 to CK8 and alphaTS1 is fully understood, mutations can be used to improve the tumor:non-tumor ratio. A scFv was made of the mab TS1 and residues earlier identified by Erlandsson et al. as important for the interaction with both its antigen CK8 and its anti-idiotype alphaTS1, were mutated to alanine or amides and expressed in E. coli. The effects of the mutations were studied by ELISA and residues important for the interactions to both CK8 and alphaTS1 were identified as mainly tyrosines, charged residues, a serine and a tryptophan. Altogether, nine amino acid residues in TS1 were found to be important in the interaction to alphaTS1 and six residues for the interaction to CK8. Important residues, clustered together in the modelled protein, were identified as residues from CDR 3 of the heavy chain and the unexpected participation of a residue in CDR 2 of the light chain. Some of the important residues are likely to be hotspots. Hotspots constitute a few residues in an interaction that contribute most to the binding, energetically. Amino acid residues in hotspots often cluster together in the center of the interaction interface, but can also be spread out to the periphery. The hotspots are often surrounded by hydrophobic patches, which are seen in the modelled TS1 protein used in this study. Amino acid residues that increased the affinity when mutated were also identified for both interactions. These residues are likely to be located outside the interacting interface. It can from this study be concluded that it is wise to precede the mutational procedure with experiments that can give guidelines for the selection of which amino acid residues to mutate. If the guidelines from the chemical modifications from Erlandsson et al. not had been used, this study would have left some residues unmutated and thereby missed important information.

Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction between Ni2+ and histidine.

Anti-beta 2-glycoprotein-I antibodies in scFv format.

Phage display was introduced almost 20 years ago. It has been used to produce large amounts of diverse proteins, to analyze protein-ligand interactions, to improve the affinity of proteins for their binding receptors, and to characterize antibody binding sites. The recombinant version of the antibody Fv is termed single-chain variable fragment (scFv). Many large phage libraries have been developed that have yielded antibodies to several hundred antigens, but only 5 human anti-beta2-glycoprotein-I and three anti-prothrombin antibodies in scFV have been so far characterized. Antibodies to beta2GP-I thus generated show 92-94% homology with their nearest germ line genes. Their mutations frequently appear to be independent of antigen. Two anti-prothrombin antibodies show strong crossreactivity with beta2GP-I. Four mouse anti-beta2GP-I scFV show less binding properties than their original counterparts, but had the same capacity of inducing experimental antiphospholipid syndrome. This pathogenicity appears to reside in the V(H)DJ(H)C(H) region of the scFv since the V(H)DJ(H)C(H) regions of pathogenic scFV combined with irrelevant V(L) J(L)C(L) regions retained their pathogenicity while the opposite failed to do so.

Neutralizing human anti-B-cell-activating factor of the TNF family (BAFF) scFv selected from phage antibody library.

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K(aff)) of 2.5 x 10(7)M(-1) without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF.

T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity.

Chimeric TCRs with an Ab-derived binding domain confer predefined specificity and MHC-independent target binding to T cells for use in adoptive immunotherapy. We investigated the impact of receptor binding affinity on the activation of grafted T cells. A series of anti-ErbB2 single-chain fragment binding domains with a K(d) ranging from 3.2 x 10(-7) to 1.5 x 10(-11) M was linked to CD3zeta-derived immunoreceptors and expressed in human PBL. Solid phase bound ErbB2 protein triggered activation of receptor-grafted T cells in a dose-dependent manner. The activation threshold inversely correlated with the affinity of the receptor binding domain. The maximum level of cellular activation, however, was the same and independent of the binding affinity. Upon binding to ErbB2(+) cells, T cells grafted with immunoreceptors carrying a single-chain fragment of K(d) < 10(-8) M were activated in a similar fashion against cells with different amounts of ErbB2 on the surface. T cells with a low affinity receptor (K(d) > 10(-8) M), however, were activated exclusively by cells with high amounts of ErbB2. In conclusion, recombinant immunoreceptors of higher affinity do not necessarily induce a more potent activation of T cells than low affinity immunoreceptors, but the higher affinity immunoreceptors exhibit less discrimination between target cells with high or low Ag expression levels.

A nonneutralizing anti-HIV-1 antibody turns into a neutralizing antibody when expressed on the surface of HIV-1-susceptible cells: a new way to fight HIV.

During HIV-1 infection or vaccination, HIV-1 envelope spikes elicit Ab responses. Neutralizing Abs block viral entry by recognizing epitopes on spikes critical for their interaction with receptor, coreceptors or fusion. In contrast, nonneutralizing Abs fail to do so because they recognize epitopes either buried or exposed but not critical for viral entry. Previously, we produced a high-affinity human mAb against the cluster II determinant of gp41. This Ab or its recombinant Fab and single-chain Fv have been repeatedly shown to bind to HIV-1 gp160 or gp41, but fail to block viral entry. We report that, surprisingly, expression of this nonneutralizing anti-HIV-1 gp41 single-chain Fv on the surface of human CD4 T cells markedly inhibits HIV-1 replication and cell-cell fusion. The inhibition targets the HIV-1 envelope at the level of viral entry, regardless of HIV-1 tropism. Although this bona fide nonneutralizing Ab does not neutralize HIV-1 entry when produced as a soluble protein, it acts as a neutralizing Ab when expressed on the cell surface. Expressing Abs on the surface of HIV-1-susceptible cells can be a new way to fight HIV-1.

Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen.

In planta expression of recombinant antibodies recognizing pathogen-specific antigens has been proposed as a strategy for crop protection. We report the expression of fusion proteins comprising a Fusarium-specific recombinant antibody linked to one of three antifungal peptides (AFPs) as a method for protecting plants against fungal diseases. A chicken-derived single-chain antibody specific to antigens displayed on the Fusarium cell surface was isolated from a pooled immunocompetent phage display library. This recombinant antibody inhibited fungal growth in vitro when fused to any of the three AFPs. Expression of the fusion proteins in transgenic Arabidopsis thaliana plants conferred high levels of protection against Fusarium oxysporum f.sp. matthiolae, whereas plants expressing either the fungus-specific antibody or AFPs alone exhibited only moderate resistance. Our results demonstrate that antibody fusion proteins may be used as effective and versatile tools for the protection of crop plants against fungal infection.

Inhibition of radiation-induced nuclear factor-kappaB activation by an anti-Ras single-chain antibody fragment: lack of involvement in radiosensitization.

We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-kappaB (NF-kappaB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-kappaB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IkappaB kinase activity and IkappaBalpha phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-kappaB. Whereas AV1Y28 inhibited NF-kappaB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-alpha-induced NF-kappaB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-kappaB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-kappaB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-kappaB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-kappaB activation. However, inhibition of NF-kappaB activation via the expression of a dominant-negative form of IkappaBalpha in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-kappaB activation, this process does not appear to play a direct role in its radiosensitizing actions.

Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain.

The mechanism of protein secretion mediated by the beta-domain of the Neisseria gonorrhoeae IgA protease, a paradigm of a family of secreted polypeptides of Gram-negative bacteria called autotransporters, has been examined using a single-chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the beta-module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N-terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S-S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N-passenger domain can be translocated through the beta-domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re-evaluation of the prevailing model for secretion of the N-domain of autotransporters.

Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus.

We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes.

Toward selection of internalizing antibodies from phage libraries.

Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).

Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells.

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

An endogenous superantigen in the rat gut lumen as a model to study the role of human protein-Fv.

Protein-Fv (pFv) is a human B superantigen which can bind the variable domain (VH) of the heavy chain of immunoglobulins (Igs) and enhance the effector functions of secretory antibodies in the gut lumen. This study describes a rat molecule related to pFv by a similar specificity to the human VH3 domain. Investigation of the content of different gut segments shows that the rat pFv is usually hidden by its binding to local Igs to form macromolecular complexes similar to the immune fortresses described in normal humans. Furthermore, the pFv level generally increases from the jejunum to the colon in parallel with the decreasing water dilution, and finally a discontinuous presence can occur along the digestive tract. Detection of this molecule in the fetus proves its non-microbial origin. Variations of the release of pFv, according to the breeding and to littermating, suggest the influence of external factors. This animal model allows the development of a study of the functions of pFv in vivo using a current laboratory species and has already provided evidence that the synthesis of this important molecule of the secretory immune system is regulated by environmental factors.

Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus.

BACKGROUND: HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children. OBJECTIVE: To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model). STUDY DESIGN: The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli. RESULTS: Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge. CONCLUSION: It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.