Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium.

Targeting a vaccine to the mucosal surface has recently been recognized as a promising approach to efficiently induce mucosal immune responses against enteric pathogens. However, poor uptake and inefficient transport of orally delivered subunit vaccines across the intestinal epithelium combined with weak immune responses still present important bottlenecks for mucosal vaccination. A possible strategy suggested to surmount these hurdles is to target the selected antigen to transcytotic receptors, such as aminopeptidase N (APN) present on enterocytes and antigen-presenting cells (APCs). Therefore, we aimed to identify potent and selective VHHs against porcine aminopeptidase N (pAPN), that were fused to the fragment crystallizable (Fc) domain of the murine IgG2a, resulting in dimeric VHH-MG fusions. Out of a library of 30 VHH-MG fusion candidates, two fusions displaying the best binding on pAPN-expressing cells were selected and showed in vivo internalization across the porcine gut epithelium. One of these fusions triggered systemic and intestinal IgA responses upon oral administration. Our results demonstrate the potential of bivalent VHH-MG fusions as delivery vehicles for vaccine antigens. VHH-mediated targeting of antigens to APN to generate protective immunity at the mucosal surface remains to be further validated.

Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization.

Simplified monomeric monoclonal antibodies consisting of a single-domain VHH, derived from camelid heavy-chain only antibodies, fused with the Fc domain of either IgG (VHH-IgG) or IgA (VHH-IgA) antibodies, are promising therapeutic proteins. These simplified single-gene encoded antibodies are much easier to manufacture and can be produced in plants and in yeast for bulk applications. These merits enable novel passive immunization applications, such as in-feed oral delivery of VHH-IgAs, which have successfully provided protection against a gastrointestinal infection in the piglet model.

Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model.

Oral antibodies that interfere with gastrointestinal targets and can be manufactured at scale are needed. Here we show that a single-gene-encoded monomeric immunoglobulin A (IgA)-like antibody, composed of camelid variable single domain antibodies (VHH) fused to IgA Fc (mVHH-IgA), prevents infection by enterotoxigenic Escherichia coli (F4-ETEC) in piglets. The mVHH-IgA can be produced in soybean seeds or secreted from the yeast Pichia pastoris, freeze- or spray-dried and orally delivered within food.

Transformation strategies for stable expression of complex hetero-multimeric proteins like secretory immunoglobulin A in plants.

Plant expression systems have proven to be exceptional in producing high-value complex polymeric proteins such as secretory IgAs (SIgAs). However, polymeric protein production requires the expression of multiple genes, which can be transformed as single or multiple T-DNA units to generate stable transgenic plant lines. Here, we evaluated four strategies to stably transform multiple genes and to obtain high expression of all components. Using the in-seed expression of a simplified secretory IgA (sSIgA) as a reference molecule, we conclude that it is better to spread the genes over two T-DNAs than to contain them in a single T-DNA, because of the presence of homologous recombination events and gene silencing. These T-DNAs can be cotransformed to obtain transgenic plants in one transformation step. However, if time permits, more transformants with high production levels of the polymeric protein can be obtained either by sequential transformation or by in-parallel transformation followed by crossing of transformants independently selected for excellent expression of the genes in each T-DNA.

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications.

Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up-to-date examples of EPT-produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.

The case for plant-made veterinary immunotherapeutics.

The excessive use of antibiotics in food animal production has contributed to resistance in pathogenic bacteria, thereby triggering regulations and consumer demands to limit their use. Alternatives for disease control are therefore required that are cost-effective and compatible with intensive production. While vaccines are widely used and effective, they are available against a minority of animal diseases, and development of novel vaccines and other immunotherapeutics is therefore needed. Production of such proteins recombinantly in plants can provide products that are effective and safe, can be orally administered with minimal processing, and are easily scalable with a relatively low capital investment. The present report thus advocates the use of plants for producing vaccines and antibodies to protect farm animals from diseases that have thus far been managed with antibiotics; and highlights recent advances in product efficacy, competitiveness, and regulatory approval.

Recombinant IgA production for mucosal passive immunization, advancing beyond the hurdles.

Vaccination is a successful strategy to proactively develop immunity to a certain pathogen, but most vaccines fail to trigger a specific immune response at the mucosal surfaces, which are the first port of entry for infectious agents. At the mucosal surfaces, the predominant immunoglobulin is secretory IgA (SIgA) that specifically neutralizes viruses and prevents bacterial colonization. Mucosal passive immunization, i.e. the application of pathogen-specific SIgAs at the mucosae, can be an effective alternative to achieve mucosal protection. However, this approach is not straightforward, mainly because SIgAs are difficult to obtain from convalescent sources, while recombinant SIgA production is challenging due to its complex structure. This review provides an overview of manufacturing difficulties presented by the unique structural diversity of SIgAs, and the innovative solutions being explored for SIgA production in mammalian and plant expression systems.

Plant expression systems for early stage discovery and development of lead therapeutic antibodies.

BACKGROUND: Antibodies for human clinical applications are predominantly produced in mammalian expression systems, with Chinese hamster ovary (CHO) cells being the gold standard. CHO cells are ideal for the manufacturing of the IgG class of antibodies, but not for the production of complex antibodies like secretory IgAs (SIgAs) and IgMs, which remain unavailable for clinical use. In contrast, plant seeds and leaves hold the promise to produce SIgAs, IgMs and similar complex antibody formats to scalable amounts. Using transient transformation of Nicotiana benthamiana leaves, complex antibody formats can be produced up to milligram amounts in less than a month. OBJECTIVE: Based on these merits, we propose a model for early-phase exploration and designing of innovative antibody formats for therapeutic application. Further in this essay, we elaborate how the model was followed during the selection of novel antibodies for seed-based production. RESULT: This exploratory model led to the engineering of novel light-chain devoid porcinized-llama antibodies (VHH-Fc) of the IgG (VHH-IgG) and IgA (VHH-IgA) isotypes and also tetravalent dimeric and SIgAs. CONCLUSION: The proposed strategy may lead to plant-based rapid engineering of innovative antibodies more apt and efficacious for therapy, and in the coarse also add to the understanding of their mode of action.

Role of plant expression systems in antibody production for passive immunization.

Passive immunization is a method to achieve immediate protection against infectious agents by administering pathogen-specific antibodies. It has proven to be lifesaving for many acute infections, and it is now also used for cancer treatment. Passive immunization therapies, however, are extremely expensive because they require large amounts of specific antibodies that are produced predominantly in mammalian expression systems. The cost for manufacturing plant-made antibodies is estimated to be comparatively low since plant production systems require relatively less capital investments. In addition, they are not prone to mammalian pathogens, which also eases downstream processing along with making it a safe expression system. Moreover, some of the recent developments in transient expression have enabled rapid, cGMP (current Good Manufacturing Practices) compliant manufacturing of antibodies. Whether lower production costs will be reflected in a lower market price for purified antibodies will be known when more plant-produced antibodies come to the market. Promisingly, the current molecular techniques in the field of in planta expression have enabled high-level production of a variety of antibodies in different plant organs, like roots/tubers/fruits, leaves and seeds, of a variety of plants, like potato, tobacco, maize, rice, tomato and pea, providing a very wide range of possible plant-based passive immunization therapies. For instance, the production of antibodies in edible tissues would allow for a unique, convenient, needle-less, oral passive immunization at the gastric mucosal surface. The technological advances, together with the innate capacity of plant tissues to assemble complex antibodies, will enable carving a niche in the antibody market. This non-exhaustive review aims to shed light on the role of plants as a flexible expression system for passive immunotherapy, which we envisage to progress alongside the conventional production platforms to manufacture specialized antibodies.

Fusion of an Fc chain to a VHH boosts the accumulation levels in Arabidopsis seeds.

Nanobodies® (VHHs) provide powerful tools in therapeutic and biotechnological applications. Nevertheless, for some applications, bivalent antibodies perform much better, and for this, an Fc chain can be fused to the VHH domain, resulting in a bivalent homodimeric VHH-Fc complex. However, the production of bivalent antibodies in Escherichia coli is rather inefficient. Therefore, we compared the production of VHH7 and VHH7-Fc as antibodies of interest in Arabidopsis seeds for detecting prostate-specific antigen (PSA), a well-known biomarker for prostate cancer in the early stages of tumour development. The influence of the signal sequence (camel versus plant) and that of the Fc chain origin (human, mouse or pig) were evaluated. The accumulation levels of VHHs were very low, with a maximum of 0.13% VHH of total soluble protein (TSP) in homozygous T3 seeds, while VHH-Fc accumulation levels were at least 10- to 100-fold higher, with a maximum of 16.25% VHH-Fc of TSP. Both the camel and plant signal peptides were efficiently cleaved off and did not affect the accumulation levels. However, the Fc chain origin strongly affected the degree of proteolysis, but only had a slight influence on the accumulation level. Analysis of the mRNA levels suggested that the low amount of VHHs produced in Arabidopsis seeds was not due to a failure in transcription, but rather to translation inefficiency, protein instability and/or degradation. Most importantly, the plant-produced VHH7 and VHH7-Fc antibodies were functional in detecting PSA and could thus be used for diagnostic applications.

Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection.

Oral feed-based passive immunization can be a promising strategy to prolong maternal lactogenic immunity against postweaning infections. Enterotoxigenic Escherichia coli (ETEC)-caused postweaning diarrhea in piglets is one such infection that may be prevented by oral passive immunization and might avert recurrent economic losses to the pig farming industry. As a proof of principle, we designed anti-ETEC antibodies by fusing variable domains of llama heavy chain-only antibodies (VHHs) against ETEC to the Fc part of a porcine immunoglobulin (IgG or IgA) and expressed them in Arabidopsis thaliana seeds. In this way, four VHH-IgG and four VHH-IgA antibodies were produced to levels of about 3% and 0.2% of seed weight, respectively. Cotransformation of VHH-IgA with the porcine joining chain and secretory component led to the production of light-chain devoid, assembled multivalent dimeric, and secretory IgA-like antibodies. In vitro analysis of all of the antibody-producing seed extracts showed inhibition of bacterial binding to porcine gut villous enterocytes. However, in the piglet feed-challenge experiment, only the piglets receiving feed containing the VHH-IgA-based antibodies (dose 20 mg/d per pig) were protected. Piglets receiving the VHH-IgA-based antibodies in the feed showed a progressive decline in shedding of bacteria, significantly lower immune responses corroborating reduced exposure to the ETEC pathogen, and a significantly higher weight gain compared with the piglets receiving VHH-IgG producing (dose 80 mg/d per pig) or wild-type seeds. These results stress the importance of the antibody format in oral passive immunization and encourage future expression of these antibodies in crop seeds.

Production of camel-like antibodies in plants.

Transgenic plants for the production of high-value recombinant complex and/or glycosylated proteins are a promising alternative for conventional systems, such as mammalian cells and bacteria. Many groups use plants as production platform for antibodies and antibody fragments. Here, we describe how bivalent camel-like antibodies can be produced in leaves and seeds. Camel-like antibodies are fusions of the antigen-binding domain of heavy chain camel antibodies (VHH) with an Fc fragment of choice. Transient expression in Nicotiana benthamiana leaves allows the production of VHH-Fc antibodies within a few days after the expression plasmid has been obtained. Generation of stable Arabidopsis thaliana transformants allows production of scalable amounts of VHH-Fc antibodies in seeds within a year. Further, we describe how the in planta-produced VHH-Fc antibodies can be quantified by Western blot analysis with Fc-specific antibodies.