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.

A two-amino acid mutation in murine IgA enables downstream processing and purification on staphylococcal superantigen-like protein 7.

With few exceptions, all currently marketed antibody therapeutics are IgG molecules. One of the reasons that other antibody isotypes are less developed are the difficulties associated with their purification. While commercial chromatography affinity resins, like staphylococcal superantigen-like 7 (SSL7) protein-containing resin, allow purification of IgAs from many animal species, these are not useful for murine IgAs. Because the mouse model is predominantly used for preclinical evaluation of IgA-based therapeutics, there is a need to develop an effective purification method for mouse IgA. Here, we adapted the sequence of a mouse IgA by mutating two amino acid residues in the fragment crystallizable (Fc) sequence to facilitate its purification on SSL7 resin. The mutated IgA Fc (hereafter referred to as IgA*) was then genetically fused to the variable domain of a llama heavy chain-only antibody (VHH) directed against the fusion protein of human respiratory syncytial virus (HRSV), resulting in VHH-IgA*, and transiently produced in infiltrated Nicotiana benthamiana leaves. These plant-produced mouse VHH-IgA* fusions were enriched by SSL7 affinity chromatography and were found to be functional in ELISA and could neutralize RSV in vitro, suggesting no detrimental effect of the mutation on their antigen-binding properties. This approach for the purification of murine IgA will facilitate downstream processing steps when designing innovative murine IgA-based fusions.

Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants.

The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

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.

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.

Evaluation of unintended effects in the composition of tomatoes expressing a human immunoglobulin A against rotavirus.

The production of neutralizing immunoglobulin A (IgA) in edible fruits as a means of oral passive immunization is a promising strategy for the inexpensive treatment of mucosal diseases. This approach is based on the assumption that the edible status remains unaltered in the immunoglobulin-expressing fruit, and therefore extensive purification is not required for mucosal delivery. However, unintended effects associated with IgA expression such as toxic secondary metabolites and protein allergens cannot be dismissed a priori and need to be investigated. This paper describes a collection of independent transgenic tomato lines expressing a neutralizing human IgA against rotavirus, a mucosal pathogen producing severe diarrhea episodes. This collection was used to evaluate possible unintended effects associated with recombinant IgA expression. A comparative analysis of protein and secondary metabolite profiles using wild type lines and other commercial varieties failed to find unsafe features significantly associated with IgA expression. Preliminary, the data indicate that formulations derived from IgA tomatoes are as safe for consumption as equivalent formulations derived from wild type tomatoes.

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants.

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (α1 and α2) with one of the two LC types (k and λ) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HCα1 and LCλ provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 µg IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.

Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules.

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop ("braid") topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.