Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose.

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.

A human anti-Pseudomonas aeruginosa serotype O6ad immunoglobulin G1 expressed in transgenic tobacco is capable of recruiting immune system effector function in vitro.

The 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 V(H) and V(L) sequences from a previously identified human IgG2 monoclonal antibody (MAb) that specifically binds to and opsonizes Pseudomonas aeruginosa serotype 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 V(H) and V(L) antigen-binding coding sequences. The plant-produced IgG1 was determined to have high-mannose glycan content and to be capable of mediating opsonophagocytosis of P. aeruginosa serotype 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.

A rAb screening method for improving the probability of identifying peptide mimotopes of carbohydrate antigens.

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.

Expression of an anti-botulinum toxin A neutralizing single-chain Fv recombinant antibody in transgenic tobacco.

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.

Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B.

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.

Immunomodulation confers herbicide resistance in plants.

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.