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Contents lists available at ScienceDirect

Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Plants as sources of natural and recombinant anti-cancer agents


J.F. Buyela,b,⁎
a
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany
b
Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074 Aachen, Germany

ARTICLEINFO ABSTRACT

Key words: Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary
Lectins metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the
Molecular farming treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines tar -
Monoclonal antibodies geting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the
Plant secondary metabolites
development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the in -
Therapeutic anti-cancer vaccines
troduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active sub-
stances, and even the production heterologous recombinant proteins, including human antibodies, lectins and
vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following
genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis
will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side
effects compared to small molecule-based drugs.

1. Introduction Society, 2010).


Cancer is not a narrowly-defined condition with a single cause, but a
1.1. Socio-economic relevance and causes of cancer group of more than 100 different diseases with shared characteristics
(American Cancer Society, 2015). Different cancers have different in-
Despite decades of medical research, cancer remains a major chal- cidences in particular demographic groups, reflecting genetic, devel-
lenge to healthcare systems (Yabroff et al., 2011). There was an in- opmental and environmental factors. For example, breast cancer is
crease from 6.2 to 8.8 million cancer-related deaths between 2003 and much more common in women than men due to developmental dif-
2015, equivalent to approximately 13% of all deaths worldwide (Bray ferences between the sexes, whereas lung cancer is more common in
et al., 2012; McGuire, 2016; Stewart and Wild, 2014; WHO, 2017). The men because more men are exposed to environmental triggers through
cancer-related mortality rate is higher in developing countries than employment or smoking. Even so, lung cancer is the most frequent
industrialized countries due to socio-economic conditions that restrict cancer-related cause of death overall, with 1.59 million cases in 2014
access to anti-cancer therapies (Sankaranarayanan, 2014). More than (American Cancer Society, 2015; McGuire, 2016).
14 million new cancer cases are reported each year, and this is expected The common characteristic shared by all types of cancer is that a
to increase by 26% over the next 35 years due to demographic changes subset of cells acquires the ability to undergo rapid and uncontrolled
and improved diagnostics (Pritzkuleit et al., 2010; Rottenberg et al., proliferation. In most cases, this is initially associated with the forma-
2010). Cancer has a devastating physiological and psychological impact tion of spatially-confined primary tumors in the affected tissue (the
on patients and their families (Faller et al., 2013; Linden and Girgis, exception being hematological malignancies, which arise from blood
2012), but on a broader level it also imposes a massive economic cells or their progenitors). Some tumors arrest at this stage and remain
burden on society, with an estimated $895.2 billion in healthcare-re- benign and non-invasive, and are not classified as cancers (Silverstein
lated payments and reduced productivity in 2008 (American Cancer et al., 2006). However, advanced tumors become progressively more

Abbreviations: APIs, active pharmaceutical ingredients; ADCC, antibody-dependent cellular cytotoxicity; ADCs, antibody–drug conjugates; ATPS, aqueous two-phase systems; CHO,
Chinese hamster ovary; CDC, complement-dependent cytotoxicity; DoE, design-of-experiments; EBV, Epstein–Barr virus; EBA, expanded-bed adsorption; FDA, Food and Drug
Administration; GMP, good manufacturing practice; HBsAg, hepatitis B soluble antigen; HBV, Hepatitis B virus; HCPs, host cell proteins; HPV, Human papillomavirus; ML1, mistletoe
lectin 1; mAbs, monoclonal antibodies; NK, natural killer; R&D, research and development; PEG, polyethylene glycol; PAT, process analytical technology; QbD, quality-by-design;
RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; T h1, T-helper; T-DNA, transfer DNA; VEGF, vascular endothelial growth factor; VFUs, vertical farming units; VLPs, virus-like
particles

Corresponding author at: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany.
E-mail addresses: johannes.buyel@rwth-aachen.de, johannes.buyel@ime.fraunhofer.de.

https://doi.org/10.1016/j.biotechadv.2018.02.002
Received 3 July 2017; Received in revised form 23 January 2018; Accepted 1 February 2018
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dysregulated and malignant: the tumor cells can not only invade ad- they are often damaged or even killed (Bernstein and Bernstein, 2015;
jacent tissues but also detach from the primary tumor, spread through Gajecka et al., 2005). The side effects of radiotherapy can therefore
the blood and/or lymphatic system, and form secondary tumors else- include the temporary depletion of hematopoietic precursor cells, and
where, a process known as metastasis (Alberts et al., 2002). Initially, six in the longer term may even induce mutations that lead to other forms
properties were proposed to define cancer: (i) self-sufficiency in growth of cancer (Berrington de Gonzalez et al., 2011; Mauch et al., 1995). The
signals, (ii) insensitivity to anti-growth signals, (iii) evasion of apop- effectiveness of radiotherapy requires a sufficient supply of oxygen
tosis, (iv) limitless replicative potential, (v) sustained angiogenesis, and because the limited direct effect of radiation on DNA is boosted by the
(vi) potential for tissue invasion and metastasis (Hanahan and generation of mutagenic free radicals when the radiation interacts with
Weinberg, 2000). Four additional characteristics have been proposed oxygen molecules. However, hypoxia often occurs in large solid tumors,
more recently: (vii) dysregulated metabolism, (viii) evasion of the im- which means that radiotherapy is less effective in this context (Harrison
mune system, (ix) genome instability, and (x) induction of inflamma- et al., 2002).
tion (Hanahan and Weinberg, 2011). However, these properties may
overemphasize the cellular aspects of the disease compared to the im- 1.2.3. Chemotherapy
pact on tissues (Sonnenschein and Soto, 2013). Chemotherapy refers to the treatment of cancer with drugs con-
The onset of cancer is often spontaneous and linked to abiotic risk taining low molecular mass active pharmaceutical ingredients (APIs),
factors such as smoking, high-energy radiation, or carcinogenic che- such as the abovementioned plant-derived mitotic inhibitor paclitaxel
mical substances, but genetic predispositions can increase the like- (Chabner and Roberts Jr, 2005). This approach is advantageous because
lihood of disease or reduce the age of onset. Biotic risk factors include the APIs can typically circulate relatively freely within body fluids,
infections with certain viruses (Cummins and Tangney, 2013; allowing them to reach tumor sites following injection into the blood,
Weinberg, 2006), such as Human papillomavirus (HPV) as the major and the drugs can also kill remote and circulating cancer cells as well as
cause of cervical cancer (Chen et al., 2015), and presumably Ep- stein– small, undetectable secondary tumors even if their precise location is
Barr virus (EBV), which is linked to Burkitt's lymphoma (Brady et al., unknown (Polireddy and Chen, 2016). Chemotherapy can also be
2007), as well as some bacterial infections, e.g. Helicobacter pylori combined with radiotherapy to increase therapeutic efficacy (Shahid,
(Hong et al., 2012). Ultimately, all factors trigger mutations and/or 2016). However, the efficacy of chemotherapy largely depends on the
epigenetic changes in the DNA structure that inactivate tumor sup- ability of the drug to penetrate tumor tissue, which is often impaired in
pressor genes such as TP53 (Bieging et al., 2014) or activate proto- large, solid tumors due to the limited vascularization (Minchinton and
oncogenes such as HER-2 (Chial, 2008). Some of the factors can im- Tannock, 2006).
mediately cause mutations including: (i) the induction of point muta- The first generation of chemotherapeutics were developed to disrupt
tions by alkylating agents, nucleoside analogs or intercalating chemi- the metabolic and/or mitotic activity of rapidly dividing cells, whereas
cals, (ii) the erroneous repair of DNA double strand breaks induced the second generation instead targeted signaling components, such as
predominantly by radiation, or (iii) the integration of foreign DNA, protein kinases and growth factor receptors (Chabner and Roberts,
disrupting the original genetic context and causing aberrant gene ex- 2005). For example, paclitaxel is a first-generation drug that disrupts
pression as observed for some viruses (Akagi et al., 2014). Other factors mitosis by preventing tubulin depolymerization, whereas gefitinib is a
act indirectly, e.g. by inducing chronic inflammation or infections that second-generation drug that inhibits signaling via the epidermal growth
promote the proliferation of a subset of cells, e.g. B-lymphocytes, in- factor receptor (Chabner and Roberts, 2005; Wani and Horwitz, 2014).
creasing the likelihood of uncontrolled growth as assumed for EBV in Some APIs used as anti-cancer agents have a simple molecular
Burkitt's lymphoma. structure facilitating their production by chemical synthesis (Neidle and
Thurston, 2005), but most of them are complex molecules that must be
1.2. Cancer therapy strategies produced using biotechnology (Baldi et al., 2008;etHaolw
., a2t014). Paclitaxel
falls into the latter category. This compound was originally isolated
Due to the heterogeneous nature of cancer there is no universal from the bark of the Pacific yew tree (Taxus brevifolia) (Wani and
treatment, but four different general approaches have evolved, which Horwitz, 2014), but is now produced in transgenic plant cell sus-
can be applied either alone or in combination (Sudhakar, 2009). pension cultures at the 75,000-L scale (Zhong, 2002). Many che-
motherapeutic agents are selective rather than specific in terms of the
1.2.1. Surgery cells they affect, i.e. some act on all rapidly dividing cells including
Surgery involves the physical removal of malignant tumor tissue, those found in tumors but also those in healthy tissues, such as hair
which in theory can provide a complete cure with a single treatment. follicle cells and B-lymphocytes. As a consequence, typical side effects
However, a small number of residual cancer cells may remain at the of chemotherapy include hair loss and a compromised immune system
excision site, eventually leading to the formation of a new tumor, or (Sfikakis et al., 2005; Trueb, 2010).
pre-malignant cells can be activated. This can be prevented by ex-
panding the excision to adjacent healthy tissue although this can have 1.2.4. Immunotherapy
severe side effects, especially in the case of brain tumors, and does not Immunotherapy harnesses the immune system in the fight against
necessarily increase the survival rate (Hernandez et al., 2009; Kubota, cancer, and is the most selective treatment approach and therefore the
2011). If metastasis has occurred, it can be difficult to locate and re- treatment associated with the least severe side effects (Caspi, 2008;
move all small secondary tumors, which is why surgery is often com- Schuster et al., 2006). Chemotherapy can be combined with im-
bined with chemotherapy or radiotherapy to increase the likelihood of munotherapy (Bang et al., 2010). The APIs used in immunotherapy are
a cure (Salama and Chmura, 2014). often proteins or peptides, including prophylactic or therapeutic vac-
cines to prevent the onset of cancer before or after exposure to biotic
1.2.2. Radiotherapy risk factors, as seen with the vaccines against HPV to prevent cervical
In radiotherapy, tumor tissue is deliberately exposed to X-rays or cancer (De Vincenzo et al., 2013; Poljak, 2012). Other strategies focus
gamma rays in order to induce DNA damage that cannot be repaired in on the introduction of cytokines to manipulate the immune response, or
rapidly-dividing cancer cells, causing them to arrest during DNA re- use antibody therapy to target cancer cells in the same way that anti-
plication and undergo apoptosis. The accuracy of radiotherapy is lim- bodies normally target pathogens (Schuster et al., 2006).
ited because ensuring that tumor cells receive a lethal dose necessarily In the latter case, monoclonal antibodies (mAbs) are directed
exposes surrounding healthy cells to lower but still harmful doses of against cancer-specific cell surface structures. Such structures can in-
radiation. DNA repair is more efficient in healthy cells but nevertheless clude receptors and other surface proteins that are overexpressed in

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Table 1
Drug types used for cancer therapy with the corresponding amounts of API required per treatment.

Drug type API Disease Treatment duration [weeks]/Number ⌀ API per patient Reference
of doses [-] (for vaccines) [mg]

Small molecule Paclitaxel Breast cancer 25 5,994 (Committee for Medicinal Products for
Human Use (CHMP), 2015a)
Vaccine Inactivated pertussis toxoid Acellular pertussis 3 11 (Thierry-Carstensen et al., 2013)
HBsAga Hepatitis B 4 440 (Aziz et al., 2006)
HBsAg Hepatitis B 3 6 (Baldy et al., 2003)
HPV L1 protein HPV infection 3 180 (McCormack, 2014)
HPV L1 protein HPV infection 3 30 (Committee for Medicinal Products for
Human Use (CHMP), 2016)
Hemagglutinin Influenza 1 9 (Kenney et al., 2004)
Hemagglutinin Influenza 1 10 (Chi et al., 2010)
Hemagglutinin Influenza 1 12 (Beran et al., 2009)
Pfb epitope fusion to HBVc core Malaria 3 35 (Nardin et al., 2004)
particles
mAb Trastuzumab Breast cancer 36-52 6,569 (Ben-Kasus et al., 2007)
Bevacizumab Colon carcinoma 240 67,140 (Committee for Medicinal Products for
Human Use (CHMP), 2015b)
Cetuximab Colon carcinoma 240 134,940 (Committee for Medicinal Products for
Human Use (CHMP), 2004)
Afliberceptd Colorectal cancer 6 2,238 (Committee for Medicinal Products for
Human Use (CHMP), 2012)
Panitumumab Colorectal cancer 6 1,902 (Committee for Medicinal Products for
Human Use (CHMP), 2007)
Rituximab Lymphoma 4 1,306 (Cheson and Leonard, 2008)
Rituximab Lymphoma 8-16 103,800 (Chames et al., 2009)
Pembrolizumab Metastatic 20 995 (Committee for Medicinal Products for
melanoma Human Use (CHMP), 2015c)

a
HBsAg – hepatitis B soluble antigen.
b
Pf – Plasmodium falciparum.
c
HBV – Hepatitis B virus.
d
This is a fusion protein used as a decoy receptor for vascular endothelial growth factor (VEGF).

tumors, or glycan structures that are more common in cancer cells. antibodies against foreign epitopes (Murphy et al., 2008), in this case
These tumor-selective targets are collectively described as tumor mar- epitopes unique to or more abundant on cancer cells or the pathogens
kers (Christiansen et al., 2014; Chung and Christianson, 2014; Duffy et that cause cancer. For example, GSK and Merck & Co. have developed
al., 2014; Weiner et al., 2012). After binding to cancer cells, the mAbs vaccines that form virus-like particles (VLPs) based on the L1 surface
can elicit antibody-dependent cellular cytotoxicity (ADCC) or protein of the HPV strains most frequently associated with cervical
complement-dependent cytotoxicity (CDC) through their constant do- cancer. The VLPs elicit an immune response against the L1 protein,
mains, causing natural killer (NK) cells to force the cancer cells into resulting in the effective clearance of infectious virus particles upon the
apoptosis (Mellstedt, 2003). Alternatively, the antibodies may block the fi
enrsctounter. In most cases this prevents the onset of a chronic HPV
binding of growth factors (Gong et al., 2004) or carry a toxic conjugate infection in women and thus reduces the risk of HPV-related cervical
such as monomethylauristatin E (Polakis, 2016), which is taken up into cancer (De Vincenzo et al., 2013; Poljak, 2012). Whereas L1-based
the tumors. Such antibody–drug conjugates (ADCs) can combine the vaccines act in a preventive manner, the HPV E6 and E7 proteins can
ADCC triggered by regular antibodies with the additional effect of a potentially be used as therapeutic vaccines, i.e. vaccines that prevent
cytotoxic effector (Peters and Brown, 2015; Scott et al., 2012). Despite the onset of cervical cancer even when a HPV infection has been es-
these benefits and several dozen approved products (Scott et al., 2012), tablished (Buyel et al., 2012; Massa et al., 2007; Venuti et al., 2009).
the pitfall of antibody therapy is the large doses of expensive and highly Lectins are another class of molecules that can be used for im-
pure mAb that are typically required for each treatment. For example, munotherapy or chemotherapy (Jiang et al., 2015). These plant-derived
doses of 3 mg per kg body mass (Ben-Kasus et al., 2007) or ~750 mg per proteins bind to various carbohydrate structures on the cell surface and
square meter of body surface area (Cheson and Leonard, 2008) can can induce immunomodulatory effects or apoptosis (Souza et al., 2013).
sometimes accumulate to an equivalent of 6–12 g per patient (Chames For example, the most abundant lectin in mistletoe (Viscum album) is
et al., 2009) (Table 1). This is why immunotherapy can cost several viscumin, also known as mistletoe lectin 1 (ML1). This is a type II ri-
thousand euros per year per patient only for the drug (not including bosome-inactivating protein (Olsnes et al., 1982) that can be used to
hospital care etc.), placing a huge financial burden on healthcare sys- treat solid tumors (Zwierzina et al., 2011) as discussed later in this
tems. The costs are exacerbated if an antibody has a short serum half- article. Most recently, nanoparticles based on native plant viruses were
life or induces an immune response in the patient, because this reduces also shown to be spectacularly successful in the treatment of solid tu-
the effective concentration of the API and increases the doses required mors (Lizotte et al., 2016).
(Glassman and Balthasar, 2014; Senter, 2009). Furthermore, the com-
parably large size of antibodies (~150 kDa) can inhibit tumor pene-
1.3. Treatment concept selection
tration and reduce the effectiveness of the treatment against large solid
tumors (Beckman et al., 2011).
Ultimately, the choice of therapeutic approach depends on the type
Anti-cancer vaccines have the potential to circumvent these pro-
and grade of the cancer, its histology and location in the body, the stage
blems because they require, in theory, only a single low-dose treatment
of the disease, and the geographical location of the patient, which often
(e.g. 15 μg) per patient, which significantly reduces the costs of pro-
determines which healthcare options are available (Manegold, 2014;
duction and administration (Table 1). Anti-cancer vaccines exploit the
Merrett, 2014). The challenging task is to decide which treatment or
ability of the human adaptive immune system to raise neutralizing
combination will most effectively clear malignant tumors from the body

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with minimal side effects, e.g. the number of cycles of chemotherapy 2. Anti-cancer agents derived from plant secondary metabolites
required (Heydarnejad et al., 2011). In this regard, individualized
medicine offers treatments that are tailored for each patient, thus in- 2.1. Natural sources of plant metabolites with anti-cancer activity
creasing therapeutic efficacy while reducing side effects. Accordingly,
this concept has attracted more interest over the last decade because Plants have been used as medicines for at least 60,000 years
implementation has become more feasible given the recent improve- (Fabricant and Farnsworth, 2001) reflecting their ability to produce
ments in cancer diagnosis, molecular biology, high-throughput cocktails of secondary metabolites with a broad range of pharmacolo-
screening, donor cell cultivation, process scale-down and single-use gical properties, including anti-cancer activity (Kuttan et al., 1997;
production systems (Klutz et al., 2015; Schilsky, 2010). Even so, per- Zarkovic et al., 1998). Phenolic compounds, such as flavonoids, are the
sonalized medicine is complex and challenging because a precise di- most promising plant-derived secondary metabolites for the treatment
agnosis is required, followed by the identification of patient-specific of cancer (Asensi et al., 2011; Wahl et al., 2011). Historically, plant-
tumor markers based on blood or biopsy samples and then the pro- derived medicines were administered orally, either by the direct con-
duction, selection or even de novo development of highly selective sumption of plant tissues or the preparation of crude extracts, which is
small-molecule anti-cancer compounds, mAbs or ADCs, and their sub- advantageous because these methods are both simple and inexpensive.
sequent application with constant monitoring for therapeutic progress Furthermore, administration is painless for the patient and safe because
(Millner and Strotman, 2016). In addition, personalized therapy would no syringe-based injection or other invasive procedures are required.
not only require a paradigm change in the way clinical trials are de- The number of publications reporting the anti-cancer activity of plant
signed today (Schork, 2015), but also a platform that can manufacture extracts is expanding rapidly: a literature search of PubMed (http://
complex APIs in comparably large amounts (gram range) in a short www.ncbi.nlm.nih.gov/pubmed) on July 1st 2017 resulted in 4753 hits
time. The latter is especially difficult to achieve with current expression including 531 review articles when using the search term “plant extract
systems, which are mostly based on cell culture, due to the lead times of anti cancer” and a massive increase in publication activity can be ob-
10–44 weeks (Fan et al., 2017). This may be unacceptable depending on served in the field since the year 2000 (Fig. 1A). It is beyond the scope
the patient's prognosis, which can involve predicted survival times of as of this review to provide an overview of all the plant-derived substances
little as ~20 weeks in the case of lung cancer (Wao et al., 2013). that have potential anti-cancer activity, their mode of action and effi-
cacy. Additionally, it is unlikely that all extracts reported thus far will
1.4. Production of anti-cancer agents be able to replicate their anti-cancer effects in larger-scale tests, which
would be necessary to make them suitable for cancer treatment stra-
Even when the specific challenges of personalized medicine are set tegies (Fritz et al., 2013; Ioannidis, 2005; Nuzzo, 2014). Furthermore,
aside, the production of sufficient quantities of high-quality anti-cancer even if the anti-cancer effects are genuine (Kwon et al., 2016), the crude
agents to meet current demand remains extremely challenging mixtures will need to be tested for safety and efficacy because, in ad-
(Siddiqui and Rajkumar, 2012). Chemical synthesis is often impractical dition to the API, they may contain metabolites with undesirable ef-
for small-molecule drugs and especially proteins due to their complex fects, and the relative concentrations of the API and other ingredients
structure, the associated low reaction yields and the unfavorable pro- would be difficult to control. For example, a raw extract of poppy
cess economics. (Papaver somniferum) seeds not only contains sanguinarine (an alkaloid
Prokaryotic expression systems are generally unsuitable for mAbs with anti-cancer activity) (Selvi et al., 2009) but also hallucinogenic
and other therapeutic proteins due to the lack of some post-transla- and addictive opiates.
tional modifications (e.g. N-linked glycosylation, which is often re- To avoid such deleterious side effects, APIs with anti-cancer activity
quired for therapeutic proteins to function properly) and the in- are usually isolated from plants or their crude extracts, allowing the
efficiency of others (e.g. disulfide bond formation, which is necessary specific mode of action to be tested at defined concentrations. As dis-
for proteins to fold correctly). The inefficient formation of disulfide cussed above, a prominent anti-cancer metabolite produced naturally
bonds causes insoluble proteins to accumulate as inclusion bodies that by plants is the taxene paclitaxel (Camidge, 2001; Wani and Horwitz,
need to be solubilized and refolded in vitro, which reduces the yield and 2014). This was approved for use in humans by the US Food and Drug
adds to the production costs (Eiberle and Jungbauer, 2010). On the Administration (FDA) in 1992. Paclitaxel is used to treat ovarian, breast
other hand, expression platforms based on mammalian cells, such as and pancreatic cancers among others (Committee for Medicinal
Chinese hamster ovary (CHO) cells, may be incompatible with the Products for Human Use (CHMP), 2015a; Wani and Horwitz, 2014) and
production of small-molecule anti-cancer drugs and proteins because in combination with gemcitabine increased the median survival time of
these are intentionally designed to be highly toxic towards mammalian patients suffering from pancreatic cancer by > 25% compared to
cells, e.g. by impairing cell division. Indeed, even reagents with limited treatment with gemcitabine alone (Von Hoff et al., 2013). Paclitaxel
toxicity would be challenging because the economics of mammalian inhibits tubulin depolymerization, thereby stabilizing the microtubule
cell systems depends on high yields, which in turn depends on opti- cytoskeleton and blocking mitosis in a concentration dependent manner
mized cell growth and high cell division rates. (Wani and Horwitz, 2014).
The following sections of this article focus on plants as either nat- Other small-molecule anti-cancer agents naturally found in plants
ural sources of anti-cancer compounds or as hosts for the production of include genistein (an isoflavone angiogenesis inhibitor), lycopene (a
recombinant anti-cancer biopharmaceutical proteins. Special emphasis carotenoid found in many fruits) and resveratrol (a stilbene found in
will be put on proteinaceous anti-cancer agents, which can exhibit an grape berries and therefore also in wines) which are currently under-
improved selectivity and reduced side effects compared to small mo- going clinical trials to test their efficacy against breast and oral cancer
lecule-based drugs. Small molecule-based anti-cancer agents have re- (Bosviel et al., 2012; King-Batoon et al., 2008; Schnekenburger et al.,
cently been reviewed elsewhere (Lalaleo et al., 2016). Plants offer all 2014; Zlotogorski et al., 2013). Additional examples of plant-derived
the functional benefits of mammalian cells because they are also higher metabolites that have shown anti-cancer activity in vitro include sev-
eukaryotic cells (i.e. protein folding and assembly, and post-transla- eral pregnane glycosides (e.g. desmiflavaside D) and alkaloids (e.g.
tional modifications) but their core processes are sufficiently distinct to mahanine) which selectively inhibit the growth of breast and prostate
allow the production of molecules that are toxic in mammals with little cancer cell lines, respectively (Jagadeesh et al., 2007; Raees et al.,
impact on the host plat cell. Some additional benefits of plants for the 2016). Anti-tumor activity has also been reported for non-psychoactive
production of anti-cancer therapeutics, such as their scalability and cannabinoids (McAllister et al., 2015) and similar activities have been
sustainability, will be discussed in concert with their merits as a plat- attributed to an increasing number of plant secondary metabolites
form for the manufacture of personalized medicines. (Schnekenburger et al., 2014).

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Fig. 1. Publication activity and anti-cancer compounds. A. Publication history for four search queries related to anti-cancer compounds derived from or produced in plants. The most
relevant query is shown as green diamonds. A Boltzmann function was fitted to each data series to illustrate the general trends in publication frequency. B. Schematic illustration of the
structures of molecules typically used for the treatment of cancer, all shown at the same scale. 1. The small molecule paclitaxel (sdf file: TA1_ideal_taxol; rcsb database; http://www.rcsb.
org) with a zoomed-in box. 2. IgG2a monoclonal antibody from mice (pdb file: 1IGT) (Harris et al., 1997). The Fc part is shown in red and green, and the Fv regions are shown in blue and
orange. 3. Therapeutic HPV vaccine candidate LicKM-E7-GGG (“3Djigsaw” (https://bmm.crick.ac.uk/~3djigsaw/) homology model using automated template selection). The HPV-E7
fusion part is shown in orange, and the lichenase part is shown in green (Buyel et al., 2012). 4. Mistletoe lectin viscumin (pdb file: 1M2T) (Krauspenhaar et al., 2002). The toxic A-chain is
shown in green, the lectin-like B-chain is shown in orange and the linking disulfide bond is highlighted by a blue arrow.

Diverse ecosystems such as tropical rainforests and coral reefs are the bark mean that expensive large-scale equipment is required. Even
thought to harbor a plethora of undiscovered molecules with similar or with optimal cultivation and processing infrastructure, the area-wise
even greater efficacy to known anti-cancer reagents, as well as yet productivity would remain low. Assuming that a 100-year-old yew tree
unknown modes of action (Mukherjee et al., 2001; Pereira et al., 2012). is covered with ~3 kg of bark containing paclitaxel at a concentration
This promising perspective is overshadowed by the massive damage of 0.14 g kg−1 (Small and Catling, 1999) and that such a tree requires
and destruction of tropical forests and reefs (van Oppen et al., 2017), 30 m2 for cultivation, the productivity is only 0.14 mg a−1 m−2. In
which is associated with the extinction of numerous species. The si- contrast, tobacco (Nicotiana tabacum) produces biomass at the rate of
tuation becomes critical when endangered plant species producing 10 kg a−1 m−2 (Stoger et al., 2002). Even if genetic engineering in to-
potential anti-cancer agents are harvested to manufacture drugs, which bacco achieves 10% of the yields in yew trees (0.014 g kg−1 biomass),
can drive them close to extinction. Precisely this scenario unfolded with the productivity is still 140 mg a−1 m−2 and thus 1000-fold higher than
the Pacific yew, which was initially the only source of paclitaxel (Wani the natural source. The production of secondary metabolites can also be
and Horwitz, 2014). This is especially relevant because large quantities increased by external factors, e.g. the lighting conditions (Buyel et al.,
of plant biomass are often required to meet demand, given the high 2015a).
doses required, e.g. ~3 g of paclitaxel per patient (Small and Catling, Small-molecule anti-cancer agents can also be produced in plant cell
1999), the miniscule amounts produced in native plants (Kelsey and cultures (Korkina and Kostyuk, 2012; Rischer et al., 2013; Tabata,
Vance, 1992), and the typically poor recovery of such compounds after 2004) typically at a scale of ~1000 L or higher (Georgiev and Weber,
extraction, which can be as low as 0.004% (Wani and Horwitz, 2014). 2014). This increases investment costs because fermentation equipment
Hence, naturally available production capacity for plant-based anti- and sterile handling are required during upstream production. How-
cancer agents tends to be insufficient to meet the demands of com- ever, cell cultures can be advantageous because it is possible to derive
mercial manufacturing. In some cases, this limitation may be cir- suspension cells directly from the native species or a close relative,
cumvented by establishing plant tissue cultures from the respective benefiting from the intrinsic metabolic capability which can optionally
medicinal plants (AbouZid, 2014; Hussain et al., 2012; Yue et al., be enhanced by genetic engineering or the addition of elicitors to in-
2016). crease yields. Such a strategy was implemented by Phyton Biotech
(Delta BC, Canada) for the production of paclitaxel in a 75,000-L scale
2.2. Production of small-molecule anti-cancer agents in genetically modified process compliant with good manufacturing practice (GMP) using
plants Taxus sp. cells to produce the API for Bristol-Myers Squibb (New York,
USA) (Zhong, 2002). The product has been marketed since 1992, with
If natural producers are not suitable for agricultural cultivation annual sales of $1.592 billion in 2000 (Exposito et al., 2009). Since
(certainly the case for the Pacific yew), the bottleneck in production then, several approaches have been pursued to increase the paclitaxel
capacity for anti-cancer reagents can be removed, and the unsustain- titers during cultivation, e.g. using elicitors such as methyl jasmonate
able depletion of natural plant populations can be avoided, if genetic (Cusido et al., 2014). This has paved the way for other anti-cancer
engineering is used either to extend existing metabolic repertoire or to agents like berberine and shikonin, which are manufactured in Coptis
introduce completely novel biosynthesis pathways into host plants that japonica and Lithospermum erythrorhizon cells, respectively (Fujita,
are compatible with large-scale manufacturing (Farre et al., 2014; 2007).
Staniek et al., 2013; Wilson and Roberts, 2012). Attributes conferring
such compatibility include high growth rates and biomass production, 3. Proteinaceous anti-cancer agents from plants
short generation times, and suitability for biomass processing
(Verpoorte et al., 2002). For example, the extraction of paclitaxel from The use of small-molecule anti-cancer agents is limited by their poor
Pacific yew trees would require a large cultivation area to grow suffi- selectivity towards cancer cells, leading to side effects caused by non-
cient trees over a period of up to 200 years (Wani and Horwitz, 2014). specific interactions with the enzymes and cell cycle components of
The trees must be logged and stripped of bark to extract paclitaxel, healthy cells. Proteins are more complex molecules (Fig. 1B) that offer
killing the trees in the process. The size of the trees and the rigidity of alternative mechanisms of action and can also be used to target cancer

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Fig. 2. Process comparison for viscumin production in E. coli and plants. A. The production of viscumin in E. coli requires individual fermentations for the two polypeptide chains of the
heterodimeric product with subsequent cell lysis, inclusion body (IB) isolation and solubilization as well as refolding in purification. B. In contrast to the ten steps in the bacterial process,
a plant-based counterpart does only consist of four stages and production is based on the full length protein.

cells more selectively than small molecules, thus reducing side effects. protein synthesis. Another study has shown that viscumin can also
stimulate T-helper (Th1) cells, thereby inducing the cellular component
3.1. Anti-cancer activity of lectins of the adaptive immune system to attack cancer cells (Zwierzina et al.,
2011).
Lectins are a heterogeneous group of glycoproteins produced by N-linked glycans are added to the viscumin B-chain as it passes
many different plant species and have probably evolved as part of the through the secretory pathway to the apoplast (Niwa et al., 2003).
molecular defense repertoire against pests and herbivores (Lannoo and These post-translational modifications are not required for its anti-
Van Damme, 2014; Van Damme, 2014; Vandenborre et al., 2011). Some tumor activity, because aglycosylated recombinant viscumin purified
lectins comprise several polypeptide chains, forming e.g. homo- from Escherichia coli inclusion bodies was also efficacious in phase I
tetramers such as ArtinM from jackfruit (Artocarpus heterophyllus) clinical trials (Zwierzina et al., 2011). However, N-linked glycosylation
(Souza et al., 2013) or heterodimers like the mistletoe lectin viscumin may increase the potency of viscumin or its serum half-life as shown for
(Kourmanova et al., 2004). The common feature of lectins is their af- human alpha-1 antitrypsin (Chung et al., 2016; Sinclair and Elliott,
finity towards carbohydrate structures, with different lectins favoring 2005). Furthermore, viscumin production in E. coli is challenging be-
different oligosaccharides. This selectivity allows some lectins to bind cause individual fermentations are required for each chain, and labor-
almost specifically to carbohydrates displayed on tumor cells, resulting ious and inefficient processing steps are necessary including the re-so-
in immunomodulatory or anti-cancer activity (Souza et al., 2013). lubilization of inclusion bodies (Eiberle and Jungbauer, 2010). The
Viscumin is the most prominent example of a plant lectin with po- subsequent co-refolding of the two chains is also inefficient and time
tential anti-cancer applications (Zwierzina et al., 2011). Viscumin is consuming. Expressing recombinant lectins in plants may therefore be
synthesized as a single polypeptide precursor which is activated by an ideal way to achieve high yields, straightforward processing and
proteolytically removing a central amino acid linker sequence. The purification, as well as a more potent APIs (Fig. 2).
resulting active form of the protein comprises two chains that are
covalently linked by a disulfide bond (Olsnes et al., 1982): the A-chain 3.2. Recombinant protein expression in plants
(former N-terminus) with N-glycosidase activity and a B-chain (former
C-terminus) which binds to carbohydrates on the cell surface (Walsh 3.2.1. Benefits and challenges
et al., 2013). The A-chain features β-sheet secondary structures but is In addition to the production of native proteins with anti-cancer
dominated by α-helices (Fig. 1B) (Krauspenhaar et al., 2002). The mode activity by some plant species, many species of plants (or the cells and
of action of the A-chain involves the cleavage of the N-glycosidic bond tissues derived from them) have been used as expression systems for
at position A4324 in the 28S rRNA of the large subunit of eukaryotic recombinant therapeutic proteins (Spiegel et al., 2016). The use of
ribosomes, hence the classification of viscumin as a type II ribosome plants for the production of recombinant proteins is known as ‘mole-
inactivating protein (Endo et al., 1988). The toxicity of purified vis- cular farming’, and to emphasize the medical relevance when these are
cumin (intravenous LD 50 in mice) is 2.4 μg kg−1 (Olsnes et al., 1982) therapeutic proteins, the alternative spelling ‘molecular pharming’ is
but doses of up to 6.4 μg kg−1 have been used to evaluate the toxic also used (Buyel, 2015; Fischer et al., 2013; Fischer et al., 1999; Ma
potential in humans during clinical phase I trials (Schoffski et al., et al., 2005; Menkhaus et al., 2004; Wilken and Nikolov, 2012). In the
2004). In comparison, the structurally-related ricin toxin from castor context of biopharmaceutical manufacturing, plants are beneficial be-
bean seeds (Ricinus communis) has a toxicity of 30 μg kg−1 (Audi et al., cause they can synthesize complex proteins with authentic post-trans-
2005). lational modifications (e.g. glycosylation, disulfide bond formation),
In contrast to the A-chain, the carbohydrate-binding B-chain of combined with low-cost upstream production, inherent process safety
viscumin is mostly composed of β-sheets (Fig. 1B) and has three intra- based on the inability of human pathogens to replicate in plants
chain disulfide bonds. Viscumin was initially shown to bind to β-ga- (Commandeur et al., 2003), and the potential for flexible and very-
lactosides, especially terminal galactose in the oligosaccharides of large-scale production (Buyel et al., 2017). The last two aspects are
glycoproteins (Gabius et al., 1992; Lee et al., 1994; Olsnes et al., 1982). particularly important when comparing plants to mammalian cells be-
However, it also binds selectively to terminal sialic acids, e.g. cause disastrous contamination with human pathogens is unlikely
IV6Neu5Ac-nLc4Cer residues in a mammalian context (Muthing et al., (Bethencourt, 2009; Zimran et al., 2011) and the manufacturing capa-
2004), especially on glycoproteins and gangliosides (Zwierzina et al., city can be rapidly adapted to meet market demands. In the future,
2011). The ganglioside CD75s carries an IV 6Neu5Ac-nLc4Cer residue intact plants could become even more attractive as expression systems
and is associated with several types of solid tumor, including pancreatic because they are sustainable, biodegradable, and allow massive process
cancer (Distler et al., 2008). The anti-tumor activity of viscumin integration, e.g. in the form of energy and/or nutrient recovery by
therefore appears to reflect the ability of the B-chain to bind selectively composting and biogas production from residual biomass.
to tumor cells, allowing the toxic A-chain to be internalized and block Two general types of expression strategies are available for plants:

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(i) transient expression using viral or bacterial vectors or combinations (His6) tag (Buyel et al., 2012). All of these modifications confer specific
thereof, and (ii) expression in transgenic plants or plant cells (Fischer properties that facilitate product purification, e.g. by two-phase ex-
and Schillberg, 2006; Paul and Ma, 2011; Twyman et al., 2003). In traction or affinity chromatography, but may not be suitable for anti-
contrast to the production of proteins in microbes and animal cells, cancer agents that are used for long-term treatment because the same
where a limited number of host systems has been standardized by the tags may be immunogenic, thus reducing therapeutic efficacy (Fischer
biomanufacturing industry (e.g. E. coli, Pichia pastoris and CHO cells) et al., 2012; Li, 2011), or their commercial used may be restricted
(The CMC Biotech Working Group, 2009), many different plant species (Conley et al., 2011). Therefore, the process-based solutions discussed
and expression strategies have been considered by academic and in- above are preferred to genetic modifications.
dustrial development teams. In terms of intact plants, tobacco and its The subsequent purification and polishing steps required to achieve
close relative N. benthamiana are now emerging as standard platforms the necessary final purity use packed-bed chromatography, but the
for the production of recombinant proteins by stable transformation product concentration can fall below 0.05 g L−1 (Menkhaus and
and transient expression, respectively, whereas cereals are regarded as Roseland, 2008) so pre-concentration by ultrafiltration/diafiltration
promising hosts for stable expression because recombinant proteins can can be prudent (Lightfoot and Moscariello, 2004; Lightfoot et al., 2008).
be stored in the seeds (Spiegel et al., 2016). Standardized platforms are Membrane absorbers can be advantageous as well due to the higher
also emerging based on tobacco, rice and carrot cell suspension cultures flow rates which reduce the overall process time (Orr et al., 2013). The
(Santos et al., 2016; Schillberg et al., 2013). Scalable unit operations use of affinity ligands (e.g. Protein A chromatography for the pur-
and more structured purification schemes are now being implemented ification of mAbs) is usually followed by orthogonal combinations of
for plant-derived products (Buyel and Fischer, 2014b, 2014d; Buyel anion and cation exchange chromatography, hydrophobic interaction
et al., 2015c). The initial extraction method for plant-derived APIs and mixed-mode chromatography, or size-exclusion chromatography
depends on the tissue and subcellular localization used for expression. (Nfor et al., 2011). For example, a two-step process consisting of Pro-
Products secreted into hydroponic medium (intact plants) or the fer- tein A and ceramic hydroxyapatite chromatography was used to isolate
mentation broth (cell culture) can be recovered directly, as in other cell a mAb from clarified tobacco extracts for phase I clinical studies (Ma
culture-based processes (Drake et al., 2009), but the concentrations et al., 2015).
may be low or the product can suffer degradation in the extracellular Integrated approaches such as expanded-bed adsorption (EBA)
environment. Recombinant proteins expressed in leaf or seed tissues are chromatography and aqueous two-phase systems (ATPS) have been
typically extracted by blade-based homogenizers/screw presses or tested for the purification of recombinant proteins from plants but were
mills, respectively (Bals and Dale, 2011; Buyel and Fischer, 2014c, either more expensive than conventional processes (Menkhaus and
2014d; Farinas et al., 2005; Hassan et al., 2014; Hassan et al., 2008; Glatz, 2005) or attracted additional regulatory scrutiny due to the
Kim et al., 2013). Aqueous buffers within the pH range 7.0–8.0 are presence of fusion tags (Reuter et al., 2014).
typically used for extraction (Buyel et al., 2015c).
3.2.3. Process design and monitoring
3.2.2. Product recovery and purification Non-platform processes designed to accommodate the properties of
During downstream processing, the process schemes rely on the individual products require time and investment, and therefore con-
same methods used in cell culture-based systems, e.g. filtration and tribute to the steadily increasing R&D costs in the biopharmaceutical
solid–liquid chromatography, but must be adapted to suit the char- industry (PhRMA, 2014). Rational protein design (Boes et al., 2011)
acteristics of plants. For example, the high particle burden (Buyel et al., and the characterization of HCPs (Buyel et al., 2013b) using open
2014) and high concentration of host cell proteins (HCPs) such as ri- source (UniProt-Consortium, 2012) and/or commercial software (Che-
bulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for proteins mical Computing Group, Montreal, Canada) can be used to establish
that were not secreted into the medium (Buyel and Fischer, 2014b) more standardized processes for plant-derived APIs. Furthermore, the
initially required adaptations that caused downstream processing to concept of quality-by-design (QbD) and its associated tools, namely
account for up to 80% of the overall process costs (Buyel et al., 2015c; design-of-experiments (DoE) and process analytical technology (PAT)
Wilken and Nikolov, 2012). Also, product concentrations below (De Beer et al., 2008; Landgrebe et al., 2010; Rathore, 2009; Rathore
0.5 g L−1 in the raw extracts (Buyel, 2015) in contrast to 5−10 g L−1 et al., 2010; Rathore et al., 2008; Read et al., 2010a; Read et al.,
achieved in cultured mammalian cells (Li et al., 2010; Shukla and 2010b), are now being integrated during process development but must
Thommes, 2010) can be challenging during initial product recovery be adapted to the large number of individual plants per batch in con-
(Winkelnkemper and Schembecker, 2010). Therefore, several pre- trast to the smaller number of fermenters in conventional processes
treatment strategies have recently been developed to facilitate clar- (D'Este et al., 2012; Juca et al., 2011; Sainz et al., 2013). For example,
ification and increase filter capacities to > 1000 L m−2, e.g. by in- new PAT monitoring has been implemented for plant cell suspension
troducing flocculants and filter aids (Buyel, 2016b; Buyel and Fischer, cultures during upstream production (Buyel et al., 2016a; Holland
2014e; Buyel et al., 2015b; Buyel et al., 2014), or by using low pH et al., 2013) and the complex mechanisms of protein expression (Buyel
(< 5.0), moderate heating (~65 °C), ultrafiltration/diafiltration and and Fischer, 2014a) and flocculation (Buyel, 2016b) have been de-
PEG precipitation to deplete HCPs by more than 90% before initial scribed using DoE, whereas mechanistic modeling was applied to the
product capture (Arfi et al., 2016; Azzoni et al., 2002; Buyel and heat treatment of leaves in the context of HCP precipitation (Buyel,
Fischer, 2014b; Buyel et al., 2016b; Menzel et al., 2016). In combina- 2016a).
tion, these strategies have the potential to mitigate the cost pressure Single-use technologies, such as filters, sensors, fermenters and
formerly attributed to downstream processing so that the costs are si- chromatography columns are becoming increasingly popular in the
milar to those incurred at the analogous stages of a mammalian cell biopharmaceutical industry (Allison and Richards, 2014; Eibl and Eibl,
culture process. 2011; Laukel et al., 2011; O'Brien et al., 2012; Shukla and Gottschalk,
A number of genetic modifications have been used to further sim- 2013; Whitford, 2010) and are likely to become an integral part of
plify the purification of proteins from clarified plant extracts. These future manufacturing (Klutz et al., 2015) because they facilitate process
modifications include oleosins as direct (Kapchie et al., 2011; Markley validation, accelerate changeover tasks, and avoid the need to clean
et al., 2006; Napier et al., 1996) or indirect fusions (McLean et al., equipment that comes into contact with the product (Fischer et al.,
2012), or the direct fusion of cereal zein proteins or similar sequences 2012). Single-use technologies are also typically implemented in plant-
(Geli et al., 1994; Torrent et al., 2009), elastin-like polypeptides based production processes (Ma et al., 2015) and the plants themselves
(Conley et al., 2009; Tian and Sun, 2011; Urry, 1988), soybean agglu- can be regarded as single-use, biodegradable bioreactors.
tinin (Tremblay et al., 2011) or the frequently used hexa-histidine Today, protein-based APIs derived from plants are still exotic

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products in the pharmaceutical industry. The only product of this class uncertainty. Although antibodies against plant glycans have been de-
currently approved as a pharmaceutical for general human use is ta- tected in human serum, to our knowledge no adverse responses against
liglucerase alfa (marketed as Elelyso) which was developed by Protalix plant-derived recombinant therapeutic proteins have been reported
Biotherapeutics and is produced in carrot cell suspension cultures (Mor, thus far (Bardor et al., 2003; Chargelegue et al., 2000; Koprivova et al.,
2015; Pastores et al., 2014). However, more than 10 further product 2004). In some cases, the non-human glycan profiles produced by
candidates including one intended for cancer therapy are already in the plants can even be advantageous, e.g. modulating the effector functions
clinical pipeline with still more in preclinical development (Gleba et al., of some plant-derived mAbs due to the carbohydrates attached to the Fc
2014). These industry-derived products combined with further APIs region, and simplifying manufacturing if glycan antenna do not need to
developed in publically funded projects (Paul et al., 2013; Sparrow be trimmed down to facilitate product uptake, as is the case for the
et al., 2007) have encouraged the regulatory authorities in the USA and enzyme glucocerebrosidase (Grabowski et al., 2014). Hence, plants can
EU to release draft guidelines for the production of plant-derived bio- be regarded not only as an alternative production system but also as a
pharmaceuticals (CPMP, 2002; FDA, 2002; Spok, 2007), filling the legal potential source of ‘biobetters’. Where authentic human-type glycosy-
vacuum that has thus far discouraged the big players in the pharma- lation is necessary for therapeutic efficacy, plants can be modified by
ceutical industry from making substantial investments in this sector mutating endogenous genes to remove residues such as core α1,3-fu-
(Das et al., 2008; Fischer et al., 2012; Ma et al., 2005). This explains the cose and β1,2-xylose (produced in plants but not mammals), and by
prominence of plant cell suspension cultures instead of intact plants introducing human genes needed for the synthesis of multi-antennary
among the first generation of approved products (Pastores et al., 2014). glycans, core α1,6-fucose, β1,4-galactose and terminal sialic acids
Plant-based production systems are now entering the biopharmaceu- (Bakker et al., 2006; Strasser, 2013, 2016; Strasser et al., 2008). These
tical arena as a competitive platform for the manufacture of bio- glyco-engineered lines can then be used as a platform for the produc-
pharmaceutical proteins, including anti-cancer antibodies, lectins and tion of recombinant proteins with authentic glycans.
vaccines.
3.4. Recombinant protein products
3.3. Transgenic plants and the potential large-scale production of anti-
cancer compounds 3.4.1. Monoclonal antibodies
Monoclonal antibodies are highly specific and efficacious APIs for
3.3.1. Scalability and product quality cancer therapy (Chiarella, 2011; Gaughan, 2015; Scott et al., 2012). In
In contrast to microorganisms and animal/plant cell cultures, 2013, a total of almost 10 tons of mAbs was manufactured with an
transgenic plants can be cultivated as intact, multi-cellular organisms. estimated market value of $US 75 billion (Ecker et al., 2015). Based on
One advantage is that sterile cultivation conditions and/or antibiotics the current forecast of a 10% annual growth rate (Research, 2013), this
are not required for upstream production, and another is that the market will cross the $US 200 billion mark before 2025. The demand
pharmaceutical crops can be grown on the same agricultural scale as for some major single products such as rituximab and etanercept is
food and feed crops, thus allowing the production of multiple tons of already about one tonne bulk API per year (Kelley, 2007). This amount
recombinant proteins to fulfil high-demand markets (Buyel et al., 2017; is likely to increase massively if the supply of these products can be
Fischer and Schillberg, 2006). For example, 179.7 million hectares of expanded to developing countries, which currently have limited access
genetically modified crops were grown in 2015, which is a 100-fold to such high-quality cancer therapeutics due to the manufacturing
increase compared to 1996 (James, 2015). Yet another advantage of costs. For example, assuming that only every second person among the
transgenic plants is that the modification is more stable than the genes 14.1 million new cancer patients every year (Pritzkuleit et al., 2010;
carried by engineered microbes or mammalian cell lines (Baneyx and Rottenberg et al., 2010) requires mAb therapy, with a typical dose of
Mujacic, 2004; Gerngross, 2004; Hellwig et al., 2004; Wurm, 2004). ~10 g of protein per patient (Chames et al., 2009), the annual demand
This ensures a defined and stable genetic status and facilitates the for mAbs related to cancer therapy will increase to 70.5 tonnes. This
generation of well-defined master and working seed banks (Fischer calculation does not take into account the increasing incidence of
et al., 2012). Transgenic lines are typically established by transforma- cancer expected in the future due to demographic changes and im-
tion using Agrobacterium tumefaciens or physical methods such as par- proved diagnostics, and also ignores the likelihood that per-patient
ticle bombardment to introduce the gene(s) of interest into the plant mAb consumption may increase due to improved survival rates.
genome, before regeneration into intact plants (Lorence and Verpoorte, Multi-tonne production scales for mAbs are difficult to achieve
2004; Newell, 2000; Rivera et al., 2012). The initial transformants are using conventional expression systems alone, even though mAb titers of
typically subjected to 3–8 breeding cycles resulting in homozygous 5−10 g L−1 are now possible on a regular basis (Li et al., 2010; Shukla
transgenic plant lines. The transformation and breeding process can and Thommes, 2010). For example, despite such high titers, the in-
take 8–24 months depending on the plant species and genetic construct, vestment costs for bioreactors would be enormous because even very-
which is comparable to the development of CHO cell lines (Fan et al., large-scale fermenters with volumes of 10,000−25,000 L (Kelley, 2007;
2017; Sack et al., 2015). Extra time may be necessary to prepare the Li et al., 2010) would only produce ~5.0 tons of mAb per year, as-
seed banks required for large-scale production (Twyman et al., 2003). suming a duration of 12 days per batch (Kelley, 2007), and would thus
Although the advent of genome editing tools such as CRISPR/Cas9 can cover less than 10% of the anticipated demand. Furthermore, the many
reduce this time (Bortesi and Fischer, 2015; Puchta and Fauser, 2014), benefits of single-use technologies are lost at such massive production
transgenic plants are probably inadequate for urgent demands such as scales because ‘disposable’ fermentation volumes are typically limited
personalized cancer therapies for patients who will not survive long in to ~2000 L and thus multiple production trains would need to operate
the absence of treatment. In contrast, transgenic plants function better simultaneously. In this scenario, a fixed stainless-steel facility would be
as a bulk production system for proteins that are required for standard more cost-effective, even taking the higher upfront costs into account
therapies in a large number of patients, e.g. anti-cancer vaccines or (Eibl et al., 2010; Shukla and Gottschalk, 2013).
mAbs such as rituximab (Buyel et al., 2017). In contrast, plants provide their own in-built single-use bioreactors,
One matter of concern when considering plants for the production and the variation in production costs is not relevant between different
of anti-cancer vaccines and antibodies is that the glycans added to production scales because plants can be numbered-up as required and
proteins by plants are structurally different to those produced in hu- the major cost factor is the additional land or greenhouse space. The per
mans, which means that injected APIs are potentially immunogenic. se high biomass yield of some crops, e.g. 10,000 tonnes a−1 km−2 for
The same limitations apply to CHO-derived mAbs, but the body of data tobacco (Stoger et al., 2002), can be increased to
available for such mAbs is several times larger, reducing the 91,000 tonnes a−1 km−2 if so called vertical farming units (VFUs) are

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used for production instead of open field cultivation (Buyel et al., 2017; (Ma et al., 2015). Furthermore, plants have been successfully used to
Holtz et al., 2015). Combining these biomass yields with antibody ex- produce griffithsin, a lectin with the potential to block the transmission
pression levels of 2 g kg−1 (Zischewski et al., 2015) and a typical mAb of Human immunodeficiency virus (HIV) (Fuqua et al., 2015; O'Keefe
recovery of 70% during purification from transgenic plants (Ma et al., et al., 2009; Vamvaka et al., 2016).
2015), implies that ~0.55 km−2 of VFU area will be sufficient to pro-
duce 70 tons of purified mAb (Buyel et al., 2017). 3.4.3. Anti-cancer vaccines
Additional benefits of VFUs include the improved containment and Vaccines are probably the most cost-effective and least invasive
more tightly controlled growth conditions compared to open-field crops approach to treat or even prevent cancer. However, in contrast to the
or greenhouses, which can be advantageous for GMP production pro- anti-cancer agents discussed above, anti-cancer vaccines are, thus far,
cesses (Fischer et al., 2012), as well as the potential for fully-automated limited to cancers triggered by biotic factors such as viruses. In theory,
handling, even in a GMP environment (Wirz et al., 2012). To achieve a single dose of microgram amounts of one or several antigens can be
the same output by fermentation, a bioreactor volume of > 350,000 L sufficient to achieve life-long protection against a specific form of
would be required. On top of the investment costs, such a setup would cancer. In practice, several doses are often required and the protection
carry an immense monetary risk given the frequency of batch failures, can decline after a few years, so boosters may be necessary (Ouattara
e.g. due to contamination (Lolas, 2013). Assuming CHO cell media costs and Laurens, 2015). Vaccines attract greater regulatory scrutiny than
of $US 55−90 L−1, each failed batch would generate losses of $US cancer therapeutics because they are typically given to healthy in-
19−32 million only for the culture medium. A more detailed discussion dividuals and the risk:benefit ratio therefore does not tolerate severe
of the benefits of plants for the very-large-scale production of mAbs can side effects, which can be acceptable in late-stage cancer patients with
be found elsewhere (Buyel et al., 2017). Once these upstream ad- no alternative (Kwok, 2011). Subunit vaccines are preferred over live or
vantages have been considered, the downstream processing and scale- attenuated pathogens because the former contain discrete antigenic
up considerations would be similar to those for cell culture-based sys- components, typically surface proteins from the pathogen, and are
tems (Ma et al., 2015; The CMC Biotech Working Group, 2009), e.g. unable to revert to virulence or replicate in the patient (Baxter, 2007).
continuous manufacturing and a preference for single-use technologies However, small protein entities tend to induce a weaker immune re-
(Angarita et al., 2015; Baur et al., 2015; Klutz et al., 2015). Plant-based sponse than larger and more complex vaccines (Bachmann and
systems may also benefit from the experiences gathered in the food Jennings, 2010). Adjuvants such as alum can be used to enhance the
processing industry in terms of large-scale processing (Goody, 1997). In immune response and thus increase protection, but VLPs are even more
summary, plants as a production platform could tap into the full po- immunogenic. In simple terms, VLPs are empty viruses, i.e. they are
tential of mAbs as anti-cancer agents for a broad range of patients in composed of typical viral structural proteins but do not carry any nu-
industrialized as well as developing countries. cleic acid cargo and therefore cannot replicate and cause disease (Wang
and Roden, 2013). The structural proteins can be produced in microbes,
3.4.2. Lectins but as discussed above the lack of post-translational modifications in
Lectins such as viscumin could potentially be developed as anti- bacteria and the high-mannose glycans produced by yeast are sig-
cancer drug candidates and a GMP-compliant process for the produc- nificant drawbacks, so higher eukaryotes are preferable. Mammalian
tion of viscumin has already been established using a standard E. coli cells produce correctly modified subunits but there is a risk of con-
expression system, allowing viscumin to be tested in clinical trials tamination with endogenous or adventitious viruses, which can ne-
(Schoffski et al., 2004; Zwierzina et al., 2011). However, the A and B cessitate the disposal of whole batches and even the closure of a facility
chains were produced by two different bacterial strains in separate (Bethencourt, 2009). To ensure that not even trace quantities of intact
fermentations and both polypeptides formed inclusion bodies, so la- or infectious viruses end up in the final drug product, virus ifisltration an
borious re-solubilization and refolding was necessary leading to poor integral part of all mammalian cell culture-based production pro-
recoveries of ~5% based on the amount of unfolded polypeptide educts, cesses. This presents a unique challenge during the production of VLPs
which is typical for such refolding processes (Eiberle and Jungbauer, because the product is typically in the same size range as any live virus
2010). Additionally, the product was not glycosylated, which may af- contaminants. Plants do not support the proliferation of human-trophic
fect its stability and efficacy, as discussed above (Li et al., 2012). Other viruses and virus filtration is not necessary (Commandeur and Twyman,
plant lectins have been expressed in the yeast P. pastoris at levels of 2005). Several VLPs have therefore been produced successfully in
6–20 mg L−1 (Lannoo et al., 2007; Oliveira et al., 2008) but glycopro- plants, including those based on HPV (Scotti and Rybicki, 2013). Plants
teins produced by yeast often bear predominantly high-mannose rather have also been used to produce subunit vaccines, e.g. a fusion protein
than complex-type glycans (Strasser, 2016). Mammalian cells are un- including a modified version of the HPV protein E7 (Buyel et al., 2012).
likely to express viscumin at high titers because the lectin would be This vaccine candidate was not only protective against cervical cancer
toxic to the host cells (Endo et al., 1988). Such issues can be overcome in the same manner as the commercial vaccines Cervarix and Gardasil,
by using plants as expression hosts because lectins are native to plants but could also be used as a therapeutic vaccine to prevent the devel-
and heterologous lectins are generally not toxic in plant systems. In the opment of cervical cancer in patients already carrying HPV (Massa
case of viscumin, the Fraunhofer Institute for Molecular Biology and et al., 2007; Venuti et al., 2009). Plants have also been used to produce
Applied Ecology IME has started to develop a plant-based expression a large number of vaccines not related to cancer, e.g. vaccines against
approach which has thus far achieved yields of ~5 mg kg −1 (our un- malaria (Spiegel et al., 2015).
published data). This is comparable to the 1.5 mg mL−1 achieved in E.
coli after refolding, but neither re-solubilization nor refolding steps are 3.4.4. Plant viruses and virus-like nanoparticles
required. Additionally, the heterodimeric viscumin is produced from a The potential of nanoparticles as carriers for the site-specific de-
single gene and the monomeric precursor is processed in a similar way livery of anti-cancer agents has attracted continued interest over several
as in the native host, which further reduces process complexity by years (van der Meel et al., 2017), but success in terms of the regulatory
avoiding the separate processes for the production and purification of approval of anti-cancer nanoparticles has been scarce, with only 104
the A and B chains. Purifying viscumin from the more complex plant polymer formulations in clinical trials compared to 2800 clinical trials
matrix is unlikely to pose a major challenge because lactosyl-Sepharose for mAbs (Venditto and Szoka, 2013). Success may have been hindered
can be used as an affinity resin, and the purification of proteins from by the size of the nanoparticles, reducing the efficiency of tumor pe-
plant extracts by affinity chromatography does not seem to be inhibited netration, but also by uncertainties about the biocompatibility and
by the larger quantity of released HCPs compared to products secreted biodegradability of nanoparticles in vivo (Lammers et al., 2012), po-
into the fermentation broth by CHO cells, as recently shown for a mAb tentially causing them to accumulate in the body with as yet unknown

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long-term effects. Most approved nanoparticles have thus far been decline afterwards (Buyel et al., 2013a), transient expression typically
synthesized from lipids or polymers (Hare et al., 2017). However, plant achieves higher product yields than transgenic plants in the short term.
virus nanoparticles based on native viruses such as Potato virus X and This is because many plant cells temporarily contain multiple copies of
Cowpea mosaic virus have been used to present HER2 epitopes as an the episomal expression vector allowing large amounts of protein to
anti-cancer vaccine (Shukla et al., 2017), or to deliver small-molecule accumulate. One potential drawback is that earlier transient expression
compounds to specific cells (Le et al., 2017), thus reducing the side studies indicated significant batch-to-batch variation in the product
effects associated with chemotherapy. These particles are proteinaceous yield (O'Neill et al., 2008), which might cause regulatory concerns in
and are thus likely to be biodegradable. Most interestingly, Cowpea terms of process reproducibility (Fischer et al., 2012). However, this
mosaic virus particles of different lengths, and without a cargo of anti- issue can be overcome by improved process parameter controls, espe-
cancer drugs, resulted in the spectacular collapse of solid tumors after 3 cially strict control of the incubation temperature and time (Buyel and
days when injected into the tumors directly (Lizotte et al., 2016). The Fischer, 2012, 2013; Buyel et al., 2013a; Larsen and Curtis, 2008). As
precise mechanism underlying this phenomenon is not yet known and discussed above, improved process control can also be achieved by
requires further investigation, but already highlights the tremendous VFUs and in fact most of the large-scale production facilities that have
potential of plant virus nanoparticles and VLPs for anti-cancer therapy. been constructed recently are built around transient expression tech-
Plants are natural hosts for plant viruses and are therefore ideal for the nologies (Holtz et al., 2015; Wirz et al., 2012). The concept has also
production of corresponding VLPs and virus coat proteins in large been successfully applied to other scenarios, such as the production of
quantities. For example, L1 protein from HPV8 was found to accumu- emergency drugs, e.g. to counter the threat of a rapidly spreading in-
late up to > 0.2 g kg−1 in N. benthamiana (Matic et al., 2012). However, fluenza pandemic (Shoji et al., 2012; Stoger et al., 2014), which has
a major bottleneck in the production of VLPs is their purification from attracted massive governmental support especially in the USA (Paul
the plant matrix, which typically relies on at least one ultra- et al., 2013). Hence, molecular pharming based on transient expression
centrifugation step (100,000 ×g), making the overall process difficult in plants combines the speed and scalability that is required to supply
to scale up. sufficient amounts of anti-cancer agents as personalized medicines in a
timely manner.
3.5. Transient expression systems as opportunities for individualized cancer
therapies 4. Conclusions

The antibodies, lectins, vaccines and VLPs discussed above are ad- The number of cancer diagnoses is likely to increase due to demo-
ministered to a large number of patients (or at-risk individuals in the graphic changes in industrialized countries along with improved and
case of vaccines) so transgenic plants are the most suitable plant-based more widely available diagnostics. Combined with growing access to
expression platform. However, mAbs can also be used as personalized modern healthcare systems in developing countries, this will increase
medicines designed to act against the unique features of a tumor in a the demand for anti-cancer agents. Biotechnological processes are
specific patient (Duffy, 2015). The demand for personalized products needed to deliver such agents, but established production systems such
will clearly be much lower than that for the general products described as those based on microbes and mammalian cell cultures are unlikely to
above, i.e. the quantities will be in the lower gram range rather than the fully satisfy the needs of this growing market due to the production
kilogram to tonne scale necessary for the most in-demand mAbs (Kelley, costs and the inherent complexity and toxicity of anti-cancer agents.
2007). However, whereas only a small number of mAbs will need to be Plants have the potential to fill this gap because they naturally produce
produced by the ton, the number of personalized products is limited a number of substances with anti-cancer activity, including small mo-
only by the number of diagnosed cancer patients. This implies that the lecules such as paclitaxel and proteins such as viscumin. This potential
regulatory approval process for personalized medicines will also need can be increased further by applying recent advances in plant bio-
to differ from conventional procedures in order to achieve feasible technology, bioprocess design and plant molecular farming to oncology
turnover times (Schork, 2015). More importantly, time is often of the products, e.g. exploiting the rapid and flexible production that can be
essence in cancer therapy, and neither conventional cell culture-based achieved using transient expression systems to manufacture in-
approaches nor transgenic plants are suitable expression systems be- dividualized medicines, or the massively-scalable production that can
cause they have lead development times of several months. In contrast be achieved using transgenic plants to manufacture bulk APIs.
to these long development cycles, transient expression in plants can be
achieved a few weeks after obtaining the DNA sequence of a persona- Acknowledgements
lized antibody, which is a much more appropriate response time
(Rosenberg et al., 2013; Shoji et al., 2012). The author acknowledges Dr. Richard M Twyman for editorial as-
Transient expression can be achieved by infecting plants with re- sistance. This work was funded by the Fraunhofer-Gesellschaft Internal
plicating viruses carrying the genes for a recombinant protein, resulting Programs under Grant No. Attract 125-600164. The author has no
in a systemic infection. This has been reported with several different conflict of interest to declare.
viruses, and personalized antibodies expressed using a Tobacco mosaic
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