Craig J. van Dolleweerd

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands.

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaPI polypeptide from S. mutans Ingbritt, which was C‐terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaPI, were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N‐terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C‐terminal region of polypeptide, inhibited AgI/II‐mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice.

Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammal; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots

The secretion of a functional, full-length monoclonal antibody complex from transgenic Nicotiana tabacum roots has been demonstrated. Initially, seeds were germinated on nitrocellulose membranes and antibody secretion detected from the developing roots. Plants were then established in hydroponic culture and secretion into the growth medium measured over 25 days. Western blotting indicated that full-length antibody was present in the medium along with other fragments. Secreted antibody was shown to be functional by binding to antigen in ELISA studies. In contrast, no antibody could be detected from transgenic Nicotiana in which the same antibody was expressed as a membrane protein in the plasmalemma. These results indicate that antibody accumulation in the growth medium is genuinely caused by rhizosecretion and not cell damage. Addition of gelatin to plant growth medium markedly increased levels of antibody accumulation. The mean antibody yield per plant was calculated to be 11.7 μg per gram root dry weight per day. Rhizosecretion may be a viable alternative to agricultural production or cell culture for the generation of monoclonal antibodies in transgenic plants. It may also give rise to novel applications for antibodies expressed in plants such as removal or neutralisation of environmental pollutants and attenuation of pathogens which infect the plant via the rhizosphere.

Passive immunity in the prevention of rabies

Prevention of clinical disease in those exposed to viral infection is an important goal of human medicine. Using rabies virus infection as an example, we discuss the advances in passive immunoprophylaxis, most notably the shift from the recommended polyclonal human or equine immunoglobulins to monoclonal antibody therapies. The first rabies-specific monoclonal antibodies are undergoing clinical trials, so passive immunisation might finally become an accessible, affordable, and routinely used part of global health practices for rabies. Coupled with an adequate supply of modern tissue-culture vaccines, replacing the less efficient and unsafe nerve-tissue-derived rabies vaccines, the burden of this disease could be substantially reduced.

Regulatory approval and a first‐in‐human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants

Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins.

Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants.

SUMMARY Monoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue. Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.

Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62‐71‐3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant‐based production system for a chimeric mouse‐human version of mAb 62‐71‐3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62‐71‐3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62‐71‐3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62‐71‐3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant‐based antibody product to benefit low‐income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer‐Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.‐C. Production, characterization, and antigen specificity of recombinant 62‐71‐3, a candidate monoclonal antibody for rabies prophylaxis in humans. FASEB J. 27, 2055–2065 (2013). www.fasebj.org

Antibody degradation in tobacco plants: a predominantly apoplastic process

BackgroundInterest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem.ResultsIn this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space.ConclusionsThe results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George’s, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Characterization of the Conformational Epitope of Guy's 13, a Monoclonal Antibody That Prevents Streptococcus mutans Colonization in Humans

ABSTRACT Guys 13 is a mouse monoclonal antibody which recognizes streptococcal antigen I/II (SA I/II), a major cell surface glycoprotein of Streptococcus mutans. In a number of clinical trials, this antibody has been shown to prevent colonization in the human oral cavity. The aim of this study was to identify the SA I/II epitope recognized by Guys 13. The data suggest that the epitope is conformational, delimited by two noncontiguous regions of the antigen: residues 45 to 457, within the N-terminal half of SA I/II, and residues 816 to 983, within the C-terminal half. In fluid-phase immunoassays a strict requirement for the simultaneous presence of both regions was demonstrated for antibody binding. Furthermore, these two regions of SA I/II were shown to have the ability to interact with each other in the absence of Guys 13 antibody, suggesting that the normal conformation of SA I/II might be determined by the interaction of these two regions.