Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.

Peptide mimotopes have been investigated as surrogate antigens of carbohydrate (CHO) targets on pathogen and tumor cells in vaccine and therapeutic discovery. One of the main bottlenecks in peptide mimotope discovery is the inability of initial screening regimes to differentiate between true mimotopes and non-mimotopes. As a result, subsequent in vivo analysis of putative peptide mimotopes is often inefficient requiring the use of experimental animals during a lengthy in vivo immunization process. Here, we demonstrate a rapid preliminary screening method to identify putative mimotopes using a recombinant antibody (rAb) library, which may increase the probability of identifying peptides that will elicit a CHO-cross-reactive response in vivo. A human naïve rAb library was screened against both an established peptide mimotope and a non-mimotope of the Group B Streptococcus (GBS) type III polysaccharide to determine if selected antibodies cross-reacted with the original GBS polysaccharide. We were able to differentiate between these two peptides because peptide-binding Abs that cross-reacted to GBS was isolated only with the peptide mimotope. We discuss the feasibility of using this method to significantly increase the breadth of screening and reduce the discovery time for peptide mimotopes.

ABSTRACTThe production of a recombinant human IgG1 in transgenic tobacco was examined to determine whether a plant-derived antibody could recruit immune system effector function against a bacterial pathogen. A plant transformation vector was engineered to contain genes for a human kappa light chain and a human gamma-1 heavy chain with VHand VLsequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizesPseudomonas aeruginosaserotype O6ad. Unique NcoI and NotI restriction sites were incorporated to flank these variable sequences, resulting in a plant transformation vector that could be engineered for expression of any other human IgG1 antibody, requiring only the substitution of other VHand VLantigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis ofP. aeruginosaserotype O6ad in vitro using human complement and human polymorphonuclear leukocytes. Thus, MAbs produced in plants from this vector could provide human IgG1 MAbs for targeting other pathogens that require the recruitment of immune system effector functions.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known and are thus classified as high-risk threats for use as bioterror agents. To examine the potential of transgenic plants as bioreactors for the production of BoNT antidotes, we transformed tobacco with an optimized, synthetic gene encoding a botulinum neurotoxin A (BoNT/A) neutralizing single-chain Fv (scFv) recombinant antibody fragment. In vitro mouse muscle twitch assays demonstrated the functional utility of this scFv extracted from tobacco for neutralizing the paralytic effects of BoNT/A at neuromuscular junctions. Based on the efficiency of the scFv capture process and the dose required to antidote a human being, 1-2 ha of this tobacco could yield up to 4 kg of scFv, which would be enough to contribute to the manufacture of 1,000,000 therapeutic doses of a monoclonal antibody (mAb) cocktail capable of neutralizing the effects of BoNT poisoning. Transgenic plants could provide an inexpensive production platform for expression of multiple mAbs toward the creation of polyclonal therapies (i.e. pooled mAbs) as the next improvement in recombinant antibody therapy.

Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole <•/<//., Science (Washington, DC), 25«:1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vìncalkaloids, and epipodophyllotoxins (C. E. Grant et al, Cancer Res., 54: 357-361,1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation charac teristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibod ies to compare MRP post-translalional modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transacted and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distri bution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these frac tions in H69AR cells may contribute to the lack of an observable accumula tion defect in these cells when compared with the transfectants.

Transgenic tobacco plants were produced that express an anti-Salmonella enterica single-chain variable fragment (scFv) antibody that binds to the lipopolysaccharide (LPS) of S. enterica Paratyphi B. The coding sequence of this scFv was optimized for expression in tobacco, synthesized and subsequently placed behind three different promoters: an enhanced tobacco constitutive ubiquitous promoter (EntCUP4), and single- and double-enhancer versions of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S). These chimeric genes were introduced into Nicotiana tabacum cv. 81V9 by Agrobacterium-mediated transformation and 50 primary transgenic (T(0)) plants per construct were produced. Among these plants, 23 were selected for the ability to express active scFv as determined by enzyme-linked immunosorbent assay (ELISA) using S. enterica LPS as antigen. Expanded bed adsorption-immobilized metal affinity chromatography (EBA-IMAC) was used to purify 41.7 mug of scFv/g from leaf tissue. Gel filtration and surface plasmon resonance (SPR) analyses demonstrated that the purified scFv was active as a dimer or higher-order multimer. In order to identify T(1) plants suitable for development of homozygous lines with heritable scFv expression, kanamycin-resistance segregation analyses were performed to determine the number of T-DNA loci in each T(0) plant, and quantitative ELISA and immunoblot analyses were used to compare expression of active and total anti-Salmonella scFv, respectively, in the T(1) generation. As S. enterica causes millions of enteric fevers and hundreds of thousands of deaths worldwide each year, large-scale production and purification of this scFv will have potential for uses in diagnosis and detection, as a therapeutic agent, and in applications such as water system purification.

Systematic research involving four chimeric gene constructions designed to express the same anti-picloram single-chain variable fragment (scFv) antibody is described. Agrobacterium-mediated transformation produced at least 25 transgenic tobacco plants with each of these, and the number of T-DNA loci in each plant was determined using kanamycin-resistance segregation assays. The relative amounts of active and total scFv in each plant were evaluated using quantitative enzyme-linked immunosorbent assay and immunoblot technologies, respectively. No significant differences in scFv activity were found among the four groups of single-locus plants, although the 35S/M construct was found to produce significantly more total anti-picloram scFv than the other three constructs. A dose-response bioassay involving T(1) seedlings from several of the highest expressers of active scFv demonstrated resistance to a constant exposure of picloram at 5 x 10(-)(8) M. Other approaches for increasing antibody-based herbicide resistance are discussed, as further improvements are needed before practical application of this technology.

In order to create a novel mechanism for herbicide resistance in plants, we expressed a single-chain antibody fragment (scFv) in tobacco with specific affinity to the auxinic herbicide picloram. Transgenic tobacco plants and seedlings expressing this scFv against picloram were protected from its effect in a dose-dependent manner. This is the first successful use of an antibody to confer in vivo resistance to a low molecular weight xenobiotic (i.e. < 1000 Da). Our results suggest the possibility for a generic antibody-based approach to create crops resistant to low molecular weight xenobiotics for subsequent use in the bioremediation of contaminated soils, crop protection and as novel selectable markers.

The catabolism of phospholipids initiated by phospholipase D (PLD, EC 3.1.4.4) is an inherent feature of developmental processes that include fruit growth and ripening. In cherry tomatoes (Lycopersicon esculentum Mill.), soluble and membrane-associated PLD activities increased during fruit development, which peaked at the mature green and orange stages. The increase in PLD activity was associated with a similar increase in the intensity of a 92 kDa band as demonstrated by western blot analysis. A full-length cDNA having 2430 bp and encoding a putative polypeptide with 809 amino acids, was isolated using tomato RNA, RT-PCR and 5' and 3' rapid amplification of cloned ends (RACE). Analysis of the primary and secondary structures showed the presence of the C2 domain, the PLD domain and several other features characteristic of PLD alpha. Microtom tomato plants transformed with antisense PLD alpha cDNA, were similar to untransformed plants and showed normal fruit set and development. The ethylene climacteric was delayed by over 7 d in the antisense PLD fruits, indicative of a slower ripening process. The leaves and unripened fruits of antisense PLD microtom plants possessed lowered PLD activity and PLD protein, as demonstrated by western blotting. However, during ripening, PLD activity in the transgenic fruits was maintained at a higher level than that in the untransformed control. Immunolocalization of PLD in microtom tomato fruits revealed the cytosol-membrane translocation of PLD during fruit development. The ripe fruits of antisense PLD celebrity plants possessed lowered PLD expression and activity and showed increased firmness and red colour. These results suggest that the expression of antisense PLD cDNA could be variable in different tomato varieties. The potential role of PLD in ethylene signal transduction events is discussed.

In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and triterpene biosynthesis and is encoded by a small gene family. The accumulation of alpha -farnesene in the skin tissue of apple fruit during storage appears to be predominantly through the classical mevalonate pathway and not through the novel glyceraldehyde-3-phosphate/pyruvate pathway independent of HMGR action. The content of a-farnesene in the skin tissue increased during the first 8 weeks of storage at 0 degreesC in air, and started declining after 12 weeks. In contrast, HMGR activity in the total membrane and soluble fractions, was the highest at the time of harvest, but decreased during the first 8 weeks in storage and remained stable thereafter. The potent ethylene action inhibitor 1-methylcyclopropene inhibited alpha -farnesene evolution and HMGR activity by 97 and 30 %, respectively. As a first step in studying the molecular mechanism of apple HMGR regulation, we have isolated and cloned a full-length cDNA (hmg1) as well as a fragment (hmg2), using apple skin mRNA and RT-PCR in the presence of degenerate oligonucleotides designed against conserved regions of plant HMGR genes. Genomic Southern analysis using probes designed for the 3'-end of the two cDNA clones confirmed the presence of at least two HMGR genes in apple. The cDNA for hmgl has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 589 residues with a relative molecular mass of 62.7 kDa. The hydropathy profile of the putative polypeptide indicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis confirmed that both hmg1 and hmg2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to C2H4, with hmg1] being expressed constitutively and hmg2 being relatively more sensitive to developmental stimuli and ethylene. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 μg/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpressMDR1. Multidrug-resistance-associated protein (MRP) andMRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. TheMRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromo some number, while the H209 cell line is hypotetraploid (4 Vi. H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vin- cristine. trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the obser vation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II«are reduced in H209/V6 cells compared to the sensitive H209 cells. Further more, the topoisomerase Ha-related protein in H209/V6 cells has an in creased electrophoretic mobility, with an apparent U, of 160,000. The levels of the topoisomerase Ila 6.1-kilobase mKNA in H209/V6 cells are reduced Ill-fold. In addition, a second topoisomerase Ila-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the W, 180,000 topoisomerase ll(! protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in 11209 cells. However, band depletion immunoblot experiments show that the topoisomerase Ha-related M, 160,000 protein in H209/V6 cells is not bound to UNA in the presence of concentrations of VP-16 that deplete the W, 170,000 topoisomerase Hu in H209 cells and the U, 180,000 topoisom erase II)! in both the resistant and sensitive cells. We conclude that quan titative and qualitative alterations in topoisomerase II«have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.