Efficacy of plant-produced recombinant antibodies against HCG.

BACKGROUND: Antibody engineering facilitates the construction of different antibody formats [single chain variable fragment (scFv), diabody, full-size chimeric monoclonal antibody] with ease. METHODS: We constructed recombinant antibodies against HCG, which is widely used in pregnancy testing and is also produced by a number of cancers. RESULTS: The recombinant antibodies were transiently expressed in tobacco leaves to levels of up to 40 mg of pure protein per kg fresh leaf weight. Enzyme linked immunosorbent assay (ELISA) and electrophoretic mobility assay (EMSA) confirmed antibody specificity for the beta subunit of beta-HCG. The efficacy was confirmed by inhibiting HCG induced testosterone production by Leydig cells in vitro and by blocking the HCG induced increase in mouse uterine weight in vivo. CONCLUSIONS: Passive immunization with recombinant HCG-specific antibodies may have clinical utility as (i) diagnostic and therapeutic tools for HCG-expressing cancers and (ii) contraceptive measures.

Specific glycosidase activity isolated from a random phage display antibody library.

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.

Optimization of scFv antibody production in transgenic plants.

BACKGROUND: Plants offer various advantages for the production of pharmaceutical proteins over conventional production systems such as bacterial or mammalian cell culture. In order to explore transgenic plants for large-scale production and storage of recombinant antibodies we tried to optimize the accumulation and stability of functionally active single chain Fv (scFv) antibodies in transgenic tobacco plants. OBJECTIVES: Two different scFv antibodies which were expressed in different plant organs and plant cell compartments have been used for the study. Accumulation levels and antibody properties such as stability and antigen-binding activity were investigated. STUDY DESIGN: For ubiquitous expression in tobacco plants, transcription of the scFv genes was controlled by the strong cauliflower mosaic virus (CaMV) 35S promoter. We used seed specific legumin B4 (LeB4) and the unknown seed protein (USP) promoters from Vicia faba for storage organ specific expression. RESULTS: High accumulation of the two different scFv proteins in transgenic tobacco plants was only achieved by retention of the recombinant antibodies in the lumen of the endoplasmic reticulum (ER). Expression levels of scFv antibodies reached up to 4-6.8% of total soluble proteins (TSP) in leaves and up to 3-4% in ripe tobacco seeds. Transgenic tobacco seeds as well as tobacco leaves facilitated stable storage of ER-accumulated scFvs over an extended (seeds) or a short (leaves) period of time. Functionally active scFv proteins could be extracted after harvesting of the leaf material--drying and storage for 1 week at room temperature. Both the amount and the binding activity of the scFv proteins remained unchanged. CONCLUSION: A plant expression system where the scFv-proteins are targeted in the ER provides not only the highest accumulation level of active single chain Fv antibodies ever reported but also a short- or long-term storage of the foreign protein in the harvested plant material.

Seed-specific immunomodulation of abscisic acid activity induces a developmental switch.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

Expression of a single-chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco.

The plant hormone abscisic acid (ABA) participates in the control of several important physiological processes in plants such as stomata regulation, seed dormancy and stress tolerance. A new strategy was developed to study these phenomena by blocking abscisic acid with intracellularly expressed specific single-chain variable fragment (scFv) antibodies. Here evidence is presented that the expression of single-chain Fv antibodies against abscisic acid in the endoplasmic reticulum of transgenic tobacco cells leads to a wilty phenotype. Stomatal conductance is increased at high CO2 concentrations dependent on the level of antibody expression in leaves. Symptoms of abscisic acid deficiency were generated in the transformants although they have even higher levels of abscisic acid than wild-type plants.