Biotechnology Advances 47 (2021) 107683

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Biotechnology Advances

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Plant-made immunotoxin building blocks: A roadmap for producing therapeutic antibody-toxin fusions

M. Knodler¨ a,b, J.F. Buyel a,b,* a Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen 52074, Germany b Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany

ARTICLE INFO ABSTRACT

Keywords: Molecular farming in plants is an emerging platform for the production of pharmaceutical proteins, and host Antibody–drug conjugate species such as tobacco are now becoming competitive with commercially established production hosts based on Chimeric fusion protein bacteria and mammalian cell lines. The range of recombinant therapeutic proteins produced in plants includes Domain linker replacement enzymes, vaccines and monoclonal antibodies (mAbs). But plants can also be used to manufacture Immunotherapy toxins, such as the mistletoe lectin viscumin, providing an opportunity to express active antibody–toxin fusion Molecular farming Oncology proteins, so-called recombinant immunotoxins (RITs). Mammalian production systems are currently used to Plant-made pharmaceutical produce antibody–drug conjugates (ADCs), which require the separate expression and purification of each Recombinant biopharmaceutical component followed by a complex and hazardous coupling procedure. In contrast, RITs made in plants are Tumor-targeted drug delivery expressed in a single step and could therefore reduce production and purificationcosts. The costs can be reduced further if subcellular compartments that accumulate large quantities of the stable protein are identified and optimal plant growth conditions are selected. In this review, we firstprovide an overview of the current state of RIT production in plants before discussing the three key components of RITs in detail. The specificity-defining domain (often an antibody) binds cancer cells, including solid tumors and hematological malignancies. The toxin provides the means to kill target cells. Toxins from different species with different modes of action can be used for this purpose. Finally, the linker spaces the two other components to ensure they adopt a stable, func­ tional conformation, and may also promote toxin release inside the cell. Given the diversity of these components, we extract broad principles that can be used as recommendations for the development of effective RITs. Future research should focus on such proteins to exploit the advantages of plants as efficient production platforms for targeted anti-cancer therapeutics.

1. Introduction target antigen making them useful for both diagnostic and therapeutic applications (Ecker et al., 2015). The therapeutic potential of some an­ Many different antigens have been identified as cellular markers of tibodies reflects their ability to inhibit or in some cases promote the solid tumors and hematological malignances (Table 1). Targeting these activity of cell-surface receptors, in addition to the general antibody- markers, and thus avoiding adverse effects, requires selective thera­ dependent cell-mediated cytotoxicity (ADCC) conferred by full-size peutic agents. Antibodies, especially monoclonal antibodies (mAbs), can mAbs with a functional Fc domain. However, the anti-tumor activity provide such selectivity for various target molecules because they of mAbs can be increased substantially by fusion with a toxic compound recognize discrete structural elements of their antigens known as epi­ to make an immunotoxin, as shown by the 22,000-fold increase in topes, reflecting their repertoire of combinatorial diversity (Ji et al., toxicity when the mAb Herceptin is converted to the immunotoxin 2019). Several mAbs may therefore bind different epitopes on the same Herceptin-Gelonin (Cao et al., 2009).

Abbreviations: ADC, antibody drug conjugate; ADCC, antibody-dependent cell-mediated cytotoxicity; BSB, binding site barrier; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; cGMP, current good manufacturing practice; CHO, Chinese hamster ovary; COG, cost of goods; DAR, drug-to-antibody ratio; FDA, Food and Drug Administration; hCFP, human cytolytic fusion protein; HCP, host cell protei; ICER, incremental cost-effectiveness ratio; mAb, monoclonal antibody; IgG, immunoglobulin G; PMP, plant-made pharmaceutical; PTM, post-translational modification; QALY, quality-adjusted life years; RIP, ribosome-inac­ tivating protein; RIT, recombinant immunotoxin; scFv, single-chain fragment variable; USP, upstream processing; VLS, vascular leak syndrome.. * Corresponding author at: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen 52074, Germany. E-mail addresses: [email protected] (M. Knodler),¨ [email protected], [email protected] (J.F. Buyel). https://doi.org/10.1016/j.biotechadv.2020.107683 Received 29 June 2020; Received in revised form 7 December 2020; Accepted 20 December 2020 Available online 27 December 2020 0734-9750/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Table 1 List of antibodies and immunotoxins targeting cell-surface antigens of solid tumors and hematological cancer cells.

Target Antibody/ligand RIT or ADCa Indication Clinical Reference phase

CD19 Anti CD19 mAb HD37-dgA B-cell lymphoma I (Stone et al., 1996) (HD37) CD20 Anti CD20 scFv MT-3724 B-cell NHL I (Fanale et al., 2018) CD22 Anti CD22 mAb Moxetumomab pasudotox Hairy cell leukemia Approved (Dhillon, 2018) CD25 Anti-Tac mAb LMB-2 Metastatic melanoma I (Kaplan et al., 2018; Powell Jr. et al., 2007) CD25 (IL-2R) IL-2 Denileukin diftitox CTCL Approved (Lansigan et al., 2010) (Ontak) CD3 Anti CD3 scFv Resimmune CTCL I (Frankel et al., 2015) CD30 Anti CD30 mAb BerH2/Saporin ALCL Pre-clinical (Pasqualucci et al., 1995) (BerH2) CD317 Anti CD317 HM1.24-ETA Multiple myeloma Pre-clinical (Staudinger et al., 2014) CD33 HuM195 HuM195/rGel AML, CML I (Borthakur et al., 2013) CD64 Anti CD64 mAb (H22, Anti CD64-ETA, H22 AML, AMML, CML Pre-clinical (Mladenov et al., 2016; Mladenov et al., 2015; m22) (scFv)-MAP Tur et al., 2011; Tur et al., 2003) CD7 Anti CD7 mAb PG001/2 AML, T-ALL Pre-clinical (Tang et al., 2016) CD74 Anti CD74 mAb 2L-Rap-hLL1 B-cell NHS Pre-clinical (Sapra et al., 2005) CD89 Anti CD89 scFV CD89-ETA AML Pre-clinical (Mladenov et al., 2015) DLL3 Rovalpituzumab Rovalpituzumab tesirine Lung cancer II (Carbone et al., 2018) EGFR TGFα TP40 Bladder cancer I (Goldberg et al., 1995; Messing and Reznikoff, 1992) EpCAM anti-EpCAM scFv Oportuzumab monatox Urothelial carcinoma II (Kowalski et al., 2012) erbB2/HER2 Anti-HER2 mAb Erb38/Kadcyla Breast carcinoma I/Approved (Montemurro et al., 2019; Wels et al., 1992) FCRL1 Anti FCRL1 mAb (E3/ E3(Fv)-PE38 B-cell NHL, CLL Pre-clinical (Du et al., 2008b) E9) FOLR1 M9346A Mirvetuximab Ovarian cancer, fallopian tube III (Moore et al., 2018) soravtansine cancer Glycoprotein 72 791T mAb Xomazyme-791 Metastatic colon cancer I (LoRusso et al., 1995) kDa IL-3R IL-3 DT388-IL-3 AML I (Frankel et al., 2008) IL-4 receptor IL-4 NBI-3001 Recurrent malignant glioma I (Weber et al., 2003) Lewis Y (BR96) BR96 mAb LMB-1(BR96 scFv-PE40) Adenocarcinoma I (Friedman et al., 1993) Mesothelin Anetumab SS1P/Anetumab Mesothelioma II (Ghafoor et al., 2018; Lambert and Morris, 2017) ravtansine Osteoactivin Glembatumumab Glembatumumab vedotin Metastatic breast cancer, II (Ott et al., 2019) (NMB) advanced melanoma Ovarian Antigen OvB3 mAb OvB3-PE Ovarian carcinoma I (Pai et al., 1991)

AML – acute myeloid leukemia; ALCL – anaplastic large-cell lymphoma; CLL – chronic lymphocytic leukemia; CTCL – cutaneous T-cell lymphoma; DLL3 – delta-like protein 3; EGFR – epidermal growth factor receptor; ETA and PE(40) – Pseudomonas exotoxin A (different abbreviations are used for the toxin in the literature); FCRL – Fc receptor like; FOLR1 – Folate receptor α; GM-CSFR – granulocyte-macrophage colony stimulation factor receptor; HER2 – human epidermal growth factor receptor 2; IL – interleukin; MAP – microtubule associated protein tau; NHL – non-Hodgkin’s lymphoma; T-ALL – T-cell acute lymphoblastic leukemia; TGF – transforming growth factor; a Italic font indicates RITs, whereas ADCs are shown in Roman.

Immunotoxins are composed of a specificity-mediating domain, immunotoxins in clinical trials from 2013 to 2017 (Beck et al., 2017; often a mAb or derivative thereof, a toxin, and a linker (Fig. 1). If the Birrer et al., 2019) with more than 100 ADCs undergoing clinical trials mAb is chemically linked to the toxin, the resulting molecule is called an as of June 2020 (Yaghoubi et al., 2020). A pre-requisite for immunotoxin antibody–drug conjugate (ADC) and the toxic payload is typically a development is the identification of tumor-associated or even tumor- small-molecule drug rather than a protein (Ricart, 2011). In contrast, specific surface markers to ensure targeted delivery, as discussed else­ recombinant immunotoxins (RITs) are polypeptides in which the anti­ where (Tolcher, 2016). body component (usually the heavy chain in the case of full-size mAbs) Here, we firstdiscuss why, despite the potential therapeutic benefits is genetically linked to a proteinaceous toxin via a contiguous poly­ of immunotoxins compared to mAbs for cancer treatment, production peptide linker. The therapeutic strategy behind both modalities is costs can be the Achilles’ heel of this novel class of biopharmaceuticals. identical, and is comparable to the “magic bullet” concept proposed by In this context, we identify the advantages of RITs compared to ADCs Paul Ehrlich, in which the selective targeting of the antibody is paired and point out how current production challenges can be overcome using with the cell-killing ability of a potent toxin (Hauner et al., 2017; plants. Next, we describe in detail the three components of RITs (spec­ Strebhardt and Ullrich, 2008). For both RITs and ADCs, targeted accu­ ificitydomain, toxin and linker) and rational selection strategies for the mulation of the toxin is achieved when the mAb binds selectively to a design of new drug candidates to be tested, ultimately in clinical trials. cell-surface marker. The latter is ideally expressed at higher levels on We conclude by extracting general principles for the design of RITs. cancer cells than healthy surrounding tissues. The internalization of RITs/ADCs by receptor-mediated endocytosis followed by the activation 2. Patient benefits and immunotoxin costs of the toxin only inside the cell results in high drug concentrations specifically in targeted cancer cells, increasing therapeutic efficacy 2.1. Benefit assessment while limiting harm to normal cells (Pirie et al., 2011). Such targeted therapies therefore have milder side effects, which is beneficial for the The global market for cancer therapeutics is expected to reach €240 treatment of vulnerable patients such as children and the elderly. Thus far, more than 1000 unique immunotoxins have been described target­ ing various types of cancer (Shan et al., 2013). The importance of immunotoxins is highlighted by the three-fold increase in the number of

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Fig. 1. Formats of antibodies and immunotoxins. Full-length mAbs (a) are commonly used as therapeutic agents, whereas disulfide-bond stabilized Fv (b), single ′ ′ chain Fv (c), Fab (e), Fab (f) or F(ab )2 (g) are truncated variants thereof. The smallest antibody derivative is a nanobody (d), consisting of only the VH segment of a mAb, typically derived from camelids. Fvs and scFvs can be fused into dimers resulting in di-scFv (h) and diabody (i) formats, which can be engineered as bispecific molecules targeting two different antigens. Antibody–drug conjugates (ADCs) are full length mAbs chemically conjugated (CL) to small-molecule drugs (blue dot) (j) or in some cases toxic proteins (red dot) (k). Recombinant immunotoxins (RITs) are genetic fusion proteins comprising a full-length mAb (l), disulfide-stabilized Fv (m), scFv (n) or Fab (o) and a toxic proteinaceous component contiguously joined in a continuous polypeptide via a potentially cleavable peptide linker (L). Theoretically, it is also possible to fuse several toxin domains in tandem (p) or to different polypeptide chains (l) to increase the drug-to-antibody ratio (DAR) of the RIT. (q) A dimeric scFv-Fc RIT that is connected to the hinge region of a full length mAb. Intermolecular disulfide bonds are shown as purple lines, VL chains in yellow, CL chains in blue, VH chains in light green, and CH chains in dark green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) billion in 20231 and more than 60 immunotoxins are currently under­ negative for health conditions that are considered worse than death going pre-clinical and clinical testing (Amani et al., 2020). These drugs (Devlin and Lorgelly, 2017; Wichmann et al., 2017). The value of Q is are urgently needed because the number of cancer patients in the USA estimated using multi-attribute utility (MAU) instruments such as the alone is expected to increase by ~30% between 2020 and 2040 (Garner EQ-5D questionnaires about different health parameters filledin by the et al., 2019). However, a careful cost-benefit assessment is required to patient (Wisløff et al., 2014). A comparison of utility scores for cancer avoid triggering the collapse of healthcare systems due to the high costs patients calculated using two different MAUs resulted in a mean of 0.785 of the new compounds. The incremental cost effectiveness ratio (ICER) is using the EQ-5D and a mean of 0.553 when using the FACT-G algorithm a quantitative measure to compare the cost effectiveness of two or more (Pickard et al., 2012). medical treatments (Hartwell et al., 2011), and is definedas the cost of a costnew coststandard treatment divided by its effectiveness (Eq. (1)) (Kaufman et al., 2017). ICER = (1) effectivnessnew effectivenessstandard Effectiveness is often based on quality-adjusted life years (QALY) (Marseille et al., 2015), a rating system that considers a quality- where the effectiveness of new and standard treatments is typically associated parameter (Q) representing the quality of life and a quanti­ measured in QALY (Eq. (2)) and only new compounds with a higher tative parameter (LY) representing years of life gained (Eq. (2)) (Wich­ effectiveness than the standard of care are considered. mann et al., 2017). The utility score Q describes the health status of the QALY = Q × LY (2) patients and is 1 for perfect health, 0 in the case of the death, and can be where Q is the utility score as a quality indicator for life and LY is the number of gained life years. Application of the ICER concept to brentuximab vedotin (Adcetris), a 1 IQVIA, Global Oncology Trends 2019, https://www.iqvia.com/insights/th CD30-specificADC for the treatment of persistent Hodgkin’s lymphoma, e-iqvia-institute/reports/global-oncology-trends-2019, accessed September 8, € -1 2020. revealed costs of ~ 280,000 QALY when used in combination with

3 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Fig. 2. Manufacturing strategies for ADCs and RITs. (a) Production regime for mAbs in CHO cells. (b) The production of ADCs requires that the three components mAb (left), toxin (middle) and linker (right) are produced and purifiedindividually before coupling, and this typically requires different platforms (mammalian cells, microbes and chemical synthesis, respectively). An intermediate purification step is necessary after the coupling procedure. (c) The production of RITs in E. coli is mostly limited to scFv-based products and may require time-consuming product refolding if the fusion protein accumulates as inclusion bodies. (d) Fully-assembled RITs can be produced in plants using a standard downstream processing scheme closely resembling the production of standard mAbs. chemotherapy as a first-line treatment (Huntington et al., 2018). For a In addition, the quantity of immunotoxins required for therapy can regular treatment regime, the costs reduced to around €90,000 QALY-1 be ~2 mg kg-1 patient body mass, with doses required every three weeks (Babashov et al., 2017), which is close to the conventional mAb tras­ in the case of brentuximab vedotin (Yi et al., 2017). Even with a + tuzumab (€110,000 QALY-1) when used for the co-treatment of HER2 restricted target population such as ~50% of all new AML diagnoses advanced gastric cancer and chemotherapy alone (Shiroiwa et al., (9000 patients) (Siegel et al., 2020), which is ~0.5% of the ~1.8 million 2011). In contrast, the median ICER for cancer-specific treatments is new cases of cancer diagnosed in the USA in 2019 (Siegel et al., 2019), only €50,000 QALY-1 (Bae and Mullins, 2014). The high ICER of novel and assuming an average body mass of 80 kg (hence 17 doses of 160 mg ADCs can thus be a hurdle for their widespread application, especially in per year) (Walpole et al., 2012), this adds up to a total demand of ~25 kg low-income countries, because it is uncertain whether health insurance of purified immunotoxin per year. Given the toxicity of these com­ providers will cover such treatments (Marseille et al., 2015). pounds, it appears questionable how such quantities can be affordably produced using established platforms. Therefore, the next two sections 2.2. Immunotoxin costs will consider (i) which types of immunotoxin are easiest to produce (fewer process steps) and can therefore help to reduce the COG and retail As for other biopharmaceuticals, the costs for immunotoxins arise in prices (Buyel and Fischer, 2017), and (ii) which product and host part to cover research and development spending including expenses for properties make it difficult to find a good match to manufacture candidates that failed during development, which can explain differ­ immunotoxins in conventional expression systems. We will then elabo­ ences between cost of goods (COG) and sales prices (Kelley, 2009). For rate further on the potential of plants. example, models for the production of therapeutic mAbs show the COG can be as low as €50–84 g-1 (Klutz et al., 2016), but retail prices of the 3. ADCs vs RITs top 15 therapeutic mAbs were ~€8000 g-1 in 2008 and the geometric mean of 46 prices for mAbs approved by the FDA in 2016 was ~€27,000 3.1. Production complexity g-1 (Hernandez et al., 2018; Kelley, 2009). However, immunotoxins are more complex to manufacture than standard mAbs (Fig. 2), for example ADCs have been tested in more than 100 clinical studies over the last due to additional coupling steps in case of ADCs. Therefore, the existing 20 years, but only seven have been approved thus far (Birrer et al., 2019; cost models are likely to underestimate the COG for this novel product Coats et al., 2019; Nessler et al., 2020). This reflectsthe immunogenicity class. of ADCs following repeated treatment cycles and their limited

4 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683 penetration through tissues into the solid tumor mass (see Section 6) activity. A single toxin unit is often linked to the heavy and/or light (Tolcher, 2016). Tumor penetration is inversely correlated to the mo­ chain of a mAb, or to the single polypeptide of a scFv, resulting in a lecular mass of an active pharmaceutical ingredient (Sun et al., 2017), defined DAR with an integer value between 1 and 4 (Francisco et al., but current ADCs typically include full-length mAbs with a narrow mass 1997a; Shan et al., 2013; Tran et al., 2013). Adding multiple copies of range of 150–170 kDa (P. Deonarain et al., 2018). There have been some the same toxin or even several different toxins in a row is possible in attempts to produce scFv-based ADCs using site-specific conjugation theory (Fig. 1p). In addition, the hydrophobicity of the peptide linker techniques such as the SNAP-tag technology (Woitok et al., 2017). and even of the toxin can be modified in a site-directed manner by However, the production of such small-format drug conjugates using protein engineering. Therefore, RITs benefitfrom greater batch-to-batch mAb fragments requires further optimization (Hoffmann et al., 2017). In reproducibility and more consistent pharmacokinetic properties that contrast, RITs can be designed in various smaller antibody-fragment can be tailored, which increases both efficacy and safety compared to formats (25–200 kDa) because only the variable regions of the anti­ ADCs (Flavell et al., 1995; Kamath and Iyer, 2015). body are required (Fig. 1). Another major drawback of ADCs is the complex manufacturing process (Fig. 2A). Some attempts have been 4. Challenges using conventional hosts for the production for made to standardize the manufacturing of non-immunogenic mAbs, RITs linkers and small molecule drugs (McCombs and Owen, 2015; Perez et al., 2014; Wang et al., 2017), but fully-assembled immunotoxins are RITs are easier to manufacture than ADCs because no chemical difficult to synthesize in mammalian cells due to the toxicity of the coupling and intermediate purification of the components is required components and the assembled product. The mAb is therefore manu­ but the product is still toxic (Fig. 2B). Since the toxin domains of RITs factured in mammalian cells whereas the toxin is generally produced by either inhibit the protein synthesis apparatus or corrupt specific struc­ chemical synthesis if it is a small-molecule drug or in microbial cells if it tures of the mitotic spindle, the production host is prone to RIT-related is a protein. The linker is generally synthetic. The three components damages. Therefore, the expression host requires a resistance towards must be separately produced and purified, then combined and coupled the toxin or should allow POI accumulation in organelles that spatially in vitro. Finally, the mature ADC must be purified again to remove un­ separate the RIT from its molecular target, which can increase product used components and other unwanted material (Ou et al., 2018). This yield. Toxin-resistant CHO cell lines are available, but RIT yields in these complex production process is expensive, which contributes to sales lines are low: for example, 0.004 g L-1 for an anti-CD3 scFv coupled to a -1 prices of ~€150,000 g for an ADC (e.g. anti-CD30 ADC Adcetris) truncated diphtheria toxin (Liu et al., 2000b; Moehring and Moehring, -1 compared to ~€27,000 g for regular mAbs (Hernandez et al., 2018). 1983) compared to 5 g L-1 as frequently reported for mAbs during fed- Ultimately, this nullifies the increased activity of ADCs compared to batch fermentation (Kelley, 2009). The production of RITs in Escher­ mAbs because the annual therapy costs per patient are ~€88,000 and ichia coli can overcome this issue, but bacteria lack the capacity for most 2 ~€86,000 respectively (Hernandez et al., 2018; Tran et al., 2013). post-translational modifications, including N-linked glycosylation, which can influence mAb behavior (Baneyx and Mujacic, 2004; Rob­ 3.2. Product homogeneity inson et al., 2015). Furthermore, mAb-based RITs are not correctly fol­ ded and assembled in bacteria and expensive multi-step refolding Even if the ADC production process is successful, the product will processes may be necessary, often starting from denatured inclusion typically exhibit not a single drug-to-antibody ratio (DAR) but a distri­ bodies (Gengenbach et al., 2019; Jungbauer and Kaar, 2007; Salehinia bution in the range of 0–8 toxin molecules per antibody due to the et al., 2018). For example, when individually expressing an antibody promiscuous nature of the conjugation chemistry and availability of heavy and light chain, both fused to Pseudomonas aeruginosa exotoxin A -1 multiple conjugation sites, which are generally the exposed side chains (ETA), in E. coli, the yield after refolding was only 0.050 g L (Hakim of particular amino acids (Sochaj et al., 2015; Tsuchikama and An, and Benhar, 2009). Even simpler, scFv-based RITs tend to form aggre­ 2018). For example, lysine residues are used in the case of Kadcyla, gates or inclusion bodies that require denaturation and refolding, hin­ whereas cysteine residues are used in the case of Adcetris (Kamath and dering the development of inexpensive large-scale production processes Iyer, 2015). A heterogeneous DAR could result in drugs with batch-to- (Chaudhary et al., 1989; Jungbauer and Kaar, 2007; Singh et al., 2015; batch or even within-batch variability and different pharmacokinetic Tran et al., 2013). The methylotrophic yeast Pichia pastoris can produce profiles (Kim et al., 2014). Recently-developed methods such as functional mAbs (Purcell et al., 2017) and has been used successfully to π-clamping allow the site-specific coupling of toxins to mAbs (Sochaj produce more than 10 toxins and derivatives thereof with titers of up to -1 et al., 2015; Zhang et al., 2016), but the IP burden of these methods 0.770 g L (Gurkan and Ellar, 2005). However, the production of RITs -1 increases the costs of manufacturing (Dai et al., 2017). Other methods resulted in low yields of only 0.037 g L (Woo et al., 2004), which may for site-specific conjugation (such as THIOMABs) depend on multi-step be linked to the release of proteases (Zuppone et al., 2019). There are a activation, which adds further processing steps and manufacturing few examples of RITs produced in insect cells, including a RIT targeting costs (Bhakta et al., 2013). These methods also lock the DAR to a specific granulocyte-macrophage colony stimulating factor (GMCSF) produced -1 value, which reduces the flexibilityin immunotoxin design and thus the in Spodoptera frugiperda, but the resulting titer was <0.001 g L (Jaha­ ability to identify molecules with an optimal therapeutic window. For nian-Najafabadi et al., 2012). Cell-free systems can be useful to screen -1 example, a DAR >4 often increases the plasma clearance of an ADC, RIT candidates because they can achieve titers of >0.250 g L for re­ reducing the circulation time and tolerability and therefore increasing combinant proteins within 24 h (Buntru et al., 2015). But the scalability toxicity, but reducing effectiveness (Kamath and Iyer, 2015). The of such systems is currently limited to about 10 L per batch, and driving force for the faster clearance rates is thought to be the increased although they do not require living cells, they still rely on toxin-sensitive hydrophobicity of ADCs compared to unconjugated mAbs (Boswell components such as ribosomes. Therefore, cell-free systems may be et al., 2011), a property which is also influenced by the linker type limited to the small-scale production of toxins that do not interfere with (Kamath and Iyer, 2015). protein synthesis, such as MAP tau. In contrast, the DAR of RITs is stable and pre-determined by the Based on the above, RITs are a therapeutically promising and inno­ engineered gene, thus achieving a reproducible pharmacological vative class of anti-cancer compounds that currently lack a suitable system for a cost-efficient, large-scale production. In the following sec­ tions we will discuss why and how plant-based systems can fillthis gap 2 Selyukh, A., Seattle Genetics cancer drug price may top $100,000, (Reuters, by offering simplifiedcandidate screening and a low cost of production Washington, DC), https://www.reuters.com/article/us-seattlegenetics-id compared to mammalian cells (Buyel and Fischer, 2017; Gengenbach USTRE77L5EB20110822, accessed September 8, 2020. et al., 2020; Nandi et al., 2016; Pilbrough et al., 2009; Rademacher et al.,

5 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Table 2 Plant-based expression of RITs and potential RIT components.

Recombinant protein Type Application Platform Product accumulation Reference

2G12 Antibody (IgG) HIV Tobacco 25 mg kg-1 (Ma et al., 2015) Anti-CD22-PE Immunotoxin ALL Chlamydomonas reinhardtii n.r. (Tran et al., 2013) BD1-G28-5 scFv Immunotoxin Non-Hodgkin’s lymphomas Tobacco cell culture n.r. (Francisco et al., 1997a) BLX-301 Antibody Non-Hodgkin’s lymphoma Duckweed n.r. (Cox et al., 2006) BR55-2 Antibody (IgG) Breast cancer Tobacco 30 mg kg-1 (Brodzik et al., 2006) CO17-1A Antibody (IgG) Colon cancer Tobacco n.r. (Verch et al., 1998) hHscFv-RC-RNase Immunotoxin Hepatocellular carcinoma Tobacco 2 mg kg-1 (Cui et al., 2012) Pr-anti-TNF Antibody (IgG) Arthritis Carrot cell culture n.r. (Rosales-Mendoza and Tello-Olea, 2015) RhinoRX Antibody (Fv) Rhinovirus prophylactic Tobacco n.r. (Paul and Ma, 2011) Trastuzumab Antibody (IgG) Breast cancer Tobacco 300 mg kg-1 (Komarova et al., 2011) VGRNb-PE Immunotoxin Inhibition of neovascularization Lactuca sativa L. 0.337 g kg-1 (Mirzaee et al., 2018) Viscumin Toxin Alternative cancer treatment Tobacco 7 mg kg-1 (Gengenbach et al., 2019)

ALL – acute lymphoblastic leukemia; HIV – human immunodeficiency virus; IgG – immunoglobulin G; scFv – single chain fragment variable; n.r. – not reported.

2019). We then elaborate on the properties of RIT components that infrastructure may currently hold back pharmaceutical companies to should be considered to produce active compounds that can be manu­ embrace the technology (Fischer and Buyel, 2020; Schillberg et al., factured at a suitable scale. 2019), but the situation may change in the near future as the potentials of the platform, especially in the context of a rapid production are being 5. Plant-made recombinant immunotoxins recognized (Capell et al., 2020; Tuse´ et al., 2020).

5.1. Recombinant protein expression in plants 5.2. Production of mAbs and toxins

All kinds of pharmaceutical proteins can be produced in plant-based Plants can produce mAbs and derivative formats like scFvs, nano­ systems such as intact transgenic plants (e.g. tobacco, rice and maize), bodies, and even more exotic constructs such as multibodies (Fig. 1) at plant cell suspension cultures (e.g. carrot and tobacco), Agrobacterium- titers of >2.0 g kg-1 wet biomass (Buyel and Fischer, 2017; Edgue et al., mediated transient expression in wild-type tobacco plants and semi-dry 2017; Zischewski et al., 2016). Furthermore, a current good artificial cell packs, as well as plant-derived cell-free lysates (Paul and manufacturing practice (cGMP)-compliant process has been developed Ma, 2011; Rademacher et al., 2019; Yao et al., 2015). This diversity, for the production of HIV-neutralizing antibodies in plants (Ma et al., combined with the fact that almost identical expression vectors can be 2015; Sack et al., 2015) and has been extensively discussed (Buyel and used in all formats, facilitates the rapid selection of host platforms that Fischer, 2014; Fischer et al., 2000; Sack et al., 2015). Also, plants have suit the properties of a specific protein product. For example, in the been used to produce small-molecule toxins for ADCs (McElroy and unlikely event that a RIT should be toxic in whole plants due to the mode Jennewein, 2018; Ou et al., 2018). More recently, tobacco plants were of action of the toxin in vivo, a rapid switch to product secretion will used for the transient expression of the lectins viscumin and ricin, which allow product recovery from plants in submersed culture (Xu and Zhang, can be used in cancer therapy (Buyel, 2018; Frigerio et al., 1998; Gen­ 2014). In addition, various vector systems such as pTRA, pEAQ and genbach et al., 2019; Sehnke et al., 1994). The ability to produce both magnICON (Gleba et al., 2005; Peyret and Lomonossoff, 2013; Rade­ mAbs and toxic proteins using the same platform in a single process macher et al., 2009), along with a plethora of untranslated regions for makes plants an ideal expression host for immunotoxins. This simple the 5’ and 3’ end of mRNAs exist that can boost expression to >4 g kg-1 approach is possible because plants produce lectins naturally, and have of wet biomass (Buyel et al., 2013; Peyret et al., 2019). High-throughput evolved to sequester them within intracellular compartments to separate screening systems are now available that facilitate the rapid testing of them from their target molecules and proteases (Frigerio et al., 1998; variant RIT candidates (Gengenbach et al., 2020; Rademacher et al., Gengenbach et al., 2019; Ma et al., 2015; Sack et al., 2015). The same 2019). mechanisms can be exploited for toxic recombinant proteins. For Plants carry out complex post-translational modifications, but dif­ example, RITs containing a ribosome-inactivating protein (RIP) should ferences in glycan structures compared to those found in humans have not be routed to the cytosol because this would interfere with host the potential to confer immunogenicity and reduce the efficacy of protein synthesis, but targeting the same RIT to the endoplasmic retic­ pharmaceutical proteins, as reported for antibodies (Faye et al., 2005; ulum (ER) avoids ribosomal contact because the polypeptide is fed co- Webster and Thomas, 2012). Even so, there is no evidence that plant translationally across the ER membrane into the lumen, and this glycans pose a risk, based the clinical testing of a replacement enzyme compartment provides access to chaperones and the glycosylation ma­ that was approved by the FDA in 2012 (Rup et al., 2017), and the ability chinery, which can increase RIT activity and stability, thus improving to control plant glycans by directing proteins to the vacuole has been yields without affecting plant growth. Furthermore, the transplastomic proven beneficial for this product because it avoids the need to trim expression and accumulation in plant chloroplasts seems to be a prom­ unwanted glycans in vitro (Tekoah et al., 2013). If authentic human ising opportunity to direct RIT accumulation into a compartment that glycosylation is required, tobacco plants can be engineered by knocking separates the product from potential molecular targets. The use of out multiple genes encoding plant glycosyltransferases and introducing chloroplast genetic engineering can result in high expression levels for those encoding the missing human transferases (Jansing et al., 2019; plant-made RITs (Table 2) at the cost of limited post-translational Strasser, 2013). Such approaches can be used in the future to generate modifications and a more time consuming process to generate trans­ plant varieties that produce less-immunogenic pharmaceutical proteins genic plant lines (Grabsztunowicz et al., 2017). Besides transplastomic compared to wild-type plants. plants, we recommend to use genome encoded RITs with chloroplast Despite these benefits,there are only few examples of plant-derived targeting signal peptides in combination with Agrobacterium-mediated recombinant biopharmaceuticals to be tested in clinical trials since the transient expression. This can reduce the time of labor during produc­ 1990’s (Ma et al., 2015; Tus´e et al., 2015). The reasons for this lack of tion and still allows the separation of active RITs from their molecular translation from bench to bedside are manifold: whereas technical and targets. The direct production of RITs with no intermediate folding or regulatory challenges that were a setback before 2010 (Fischer et al., assembly steps reduces the complexity and cost of downstream pro­ 2012) have largely been resolved, a lack of large-scale manufacturing cessing, making the process far simpler and less prone to failure

6 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683 compared to the production of ADCs and to the production of RITs in glycine-serine linker for the fusion of one ETA molecule per scFv other platforms, including E. coli (Fig. 2C) (Gengenbach et al., 2019). (DAR=2) (Fig. 1q). Both RITs accumulated in the algal chloroplasts as Despite the advantages and the many types of recombinant proteins that soluble and functional proteins with expression levels of 0.2-0.4% of the have been produced in plants or plant cells (Spiegel et al., 2018), we are total soluble protein in the lysate (Tran et al., 2013). Both variants were aware of only four RITs that have been expressed in plant-based systems extracted by sonication lysis followed by high-speed centrifugation and and these are discussed in turn below. anti-FLAG M2 affinitychromatography to capture the N-terminal FLAG tag. The yield and purity were not reported. The enzymatic activity of ETA was confirmedin vitro, and cell-based 5.3. Bryodin-1 single-chain immunotoxin produced in plant cells + assays indicated specificcytotoxicity against CD22 cell lines with IC50 -1 The toxic RIP bryodin-1 (BD1) was fused to a scFv derived from the values of 19–104 μg L (250–1390 pM) for the monomeric immunotoxin -1 anti-CD40 antibody G28-5 (Francisco et al., 1997a). A secreted version and 1–4 μg L (11–42 pM) for its divalent counterpart (Tran et al., of BD1-G28-5 was produced in tobacco (Nicotiana tabacum cv. NT1) 2013). The divalent construct was therefore 22–33 times more effective, suspension cells, and a purity of >90% was achieved following cation potentially due to the presence of two antigen binding sites combined exchange chromatography and affinity chromatography with a with the higher DAR. Such synergistic effects should be investigated in customized resin featuring an immobilized CD40 ligand (Francisco future RITs, for example by using full-length mAbs with two or more et al., 1997a). This affinityresin was chosen because scFv RITs lack the toxic payloads (DAR≥2). A strong inhibition of tumor growth was re­ antibody Fc and Fab regions that are allow the selective purification of ported in a xenograft mouse model, confirming the efficacy of RITs antibodies using Protein A or Protein G, respectively. Since 2012, Pro­ produced in the algal expression platform (Lozano, 2012). tein L resins have become commercially available that facilitate the purification of RITs that contain a kappa light chain as part of the 5.5. Expression of an ETA-based immunotoxin in transplastomic lettuce specificitymediating domain. Other RITs can still be purifiedby easy-to- manufacture custom resins using linear epitopes of the target antigen to The RIT VGRNb-PE consisting of ETA fused to the C-terminus of a capture RITs with a correctly-folded antigen-binding domain (Knodler¨ codon optimized VEGFR2-specific nanobody (3VGR19) was expressed et al., 2019b; Rühl et al., 2018). In any case, a second orthogonal in the chloroplasts of transgenic lettuce (Lactuca sativa L.) (Mirzaee chromatography step could use ligands that capture the toxin compo­ et al., 2018). The presence and correct assembly of the 57 kDa VGRNb- -1 nent. The expression level of BD1-G28-5 in plant cells was not reported, PE protein was confirmedby western blot and the titer was 0.337 g kg although the yield of BD1 alone was only 0.030 g L-1 (Francisco et al., leaf tissue (equivalent to a liter of fermentation volume (Gengenbach 1997a). et al., 2019)) (Mirzaee et al., 2018). The yield and purity after immo­ During initial in vitro assays, BD1-G28-5 showed no toxicity towards bilized metal affinity chromatography purification were not reported. – + + the CD40 control cell line HPB-ALL or the CD40 cell lines Daudi, T51 The purified VGRNb-PE was toxic towards the VEGFR2 cell line -1 or Raji, which lack an efficient receptor-mediated internalization 293KDR with an IC50 of ~10 μg L (~170 pM) whereas there was no mechanism (Francisco et al., 1997a; Francisco et al., 1997b). In contrast, effect against HEK293 control cells at RIT concentrations of up to 100 μg + -1 BD1-G28-5 was specifically toxic towards CD40 non-Hodgkin’s lym­ L (Mirzaee et al., 2018). phoma cells with functional receptor-mediated internalization, showing -1 a half maximal inhibitory concentration (IC50) of 0.2–1.0 μg L (3~19 5.6. Production of hHscFv-RC-RNase in tobacco pM). In comparison, the CD33-specific ADC Mylotarg achieved an IC50 of 2–24 μg L-1 (13~155 pM) against HL-60 cells (Amico et al., 2003; The scFv-RC-RNase fusion protein consists of the human scFv HAb25 Yamauchi et al., 2014), and the CD30-specific ADC Adcetris achieved and the cytotoxic ribonuclease RC-RNase from the American bullfrog -1 3 IC50 values <10 μg L (10~100 pM) against AML cell lines (Chen et al., Rana catesbeiana (Cui et al., 2012). The fusion protein was produced in -1 2013a). The CD19-specificADCs huB4-DGN462 and SAR3419 achieved transgenic tobacco plants with a yield of only up to 2.0 mg kg fresh leaf -1 -1 median IC50 values of ~16 μg L (100 pM) and ~5660 μg L (37,000 biomass. The toxicity of unpurified scFv-RC-RNase was tested against pM), respectively, against different AML cell lines (Hicks et al., 2019). human hepatoma cell lines SMMC7721 and HEPG2, and resulted in IC50 -1 Only CD33-specific ADC IMGN779 achieved IC50 values that were values of 90 and 110 μg L (2000 and 2400 pM), respectively. similar to those of the plant-derived RIT when tested against AML cell In summary, mAbs and toxins can be produced in plants with cGMP- lines, but there was a much broader range of values (0.306~459 μg L-1; compliant processes in place. A first set of RITs has been successfully 2–3000 pM) and the average was higher (Whiteman et al., 2014). This produced in plants with IC50-based activities comparable to ADCs. The lower average toxicity may reflect the inefficient tumor penetration of RIT yields in plants exceeded those achieved using other host systems in ADCs due to their mass, which is ~3-fold higher compared to scFv-based several cases but require to be further improved to reach accumulation RIT (~152 vs ~55 kDa). The RIT carries a single toxin on every molecule levels of other non-toxic recombinant proteins. The accumulation levels (DAR=1) whereas the ADCs Mylotarg and Adcetris have a DAR >1, but reported for plant-made RITs are rather at the lower end of the given -1 the intrinsic toxicity of the toxins may differ. range of 2-337 mg kg . However, the expression levels in planta and more so the purity and yield after purificationare often not reported in 5.4. Anti-CD22 exotoxin A immunotoxin produced in algal chloroplasts detail (Table 2) and unit operations with limited scalability and unclear compatibility with cGMP production are used in many studies. These An antibody targeting the CD22 B-cell surface epitope was geneti­ limitations must be addressed in the future because they allow only a cally fused to ETA domains II and III and expressed in the alga Chla­ rough estimate of RIT production costs in plants. Assuming RIT levels of -1 mydomonas reinhardtii (Tran et al., 2013). The CD22-specific RIT was 13 mg kg as described for viscumin transiently expressed in tobacco tested in two formats: a standard scFv fusion protein (DAR=1) and a plants (Gengenbach et al., 2019) with recoveries after purification of dimeric scFv construct linking the variable domains of the IgG1 heavy 80% and process costs of ~€250 per kg plant biomass (Buyel, 2019), the -1 and light chains to the hinge and constant domains of an IgG1 using a COG for a purifiedplant-derived RIT would be ~€25,000 g . This would leave between €125,000 g-1 and €1,575,000 g-1 to cover for R&D and marketing costs and to generate profit,when compared the sales prices 3 European Medicines Agency, Committee for Medicinal Products for Human of brentuximab vedotin (Adcetris) and gemtuzumab ozogamicin Use (CHMP), Assessment Report: Adcetris (EMEA/H/C/002455), https://www. (Mylotarg), respectively (Selby et al., 2019). The most promising way to ema.europa.eu/en/documents/assessment-report/adcetris-epar-public-assess improve the economic competitiveness of plants as a production plat­ ment-report_en.pdf, accessed September 8, 2020. form is to increase accumulation levels. An aspect that has largely been

7 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Table 3 Proteinaceous toxins as components of immunotoxins including their origin, molecular mass, biochemical mechanism of action and development stage as the toxin payload of immunotoxins. Toxin Origin ~Size Biochemical mechanism Clinical Reference [kDa] phase

Abrin Abrus precatorius 65 Type-II RIP Pre-clinical (Gadadhar and Karande, 2013) Angiogenin Homo sapiens 14 RNase Pre-clinical (Cong et al., 2016; Stocker et al., 2003) Bouganin Bougainvillea spectabilis 30 Type-I RIP Phase I (Bortolotti et al., 2018; Entwistle et al., 2013) Colocin 1 Citrullus colocynthis 30 Type-I RIP Pre-clinical (Bolognesi et al., 1990; Dosio et al., 1996) Cytolysin A Salmonella typhi 34 Actinoporin Pre-clinical (Mutter et al., 2018) Diphtheria toxin Corynebacterium 62 ADP ribosylation of the diphthamide Phase II (Kreitman and Pastan, 1998; Selim et al., 2016) diphtheriae residue in EF-2 Ebulin I Sambucus ebulus 56 Type-II RIP Pre-clinical (Citores et al., 2002; Ferreras et al., 2011; Jimenez´ et al., 2015) Exotoxin A Pseudomonas aeruginosa 38 ADP-ribosylation of the diphthamide Approved (Dhillon, 2018; Moss et al., 2019) residue in EF-2 Fragaceatoxin C Actinia fragacea 20 Actinoporin Pre-clinical (Mutter et al., 2018) Gelonin Gelonium multiflorum 30 Type-I RIP Pre-clinical (Badr et al., 2014) Granzyme B Homo sapiens 34 Serine protease Pre-clinical (Stahnke et al., 2008) MAP tau Homo sapiens 40a Microtubule stabilizing protein Pre-clinical (Hristodorov et al., 2013) Modeccin Adenia digitata 63 Type-II RIP None (Olsnes et al., 1978) Moschatin Cucurbita moschata 29 Type-I RIP Pre-clinical (Chuan Xia et al., 2003) Pokeweed antiviral Phytolacca americana 27 Type-I RIP Pre-clinical (Myers et al., 1991) protein Ricin Ricinus communis 63 Type-II RIP Phase III (Barenholz, 2012) Saporin Saponaria officinalis 30 Type-I RIP Pre-clinical (Bergamaschi et al., 1996; Giansanti et al., 2018; Günhan et al., 2008) Sechiumin Sechium edule 27 Type-I RIP None (Wu et al., 1998) Trichosanthin Trichosanthes kirilowii 27 Type-I RIP Pre-clinical (Li et al., 2010; Zhao et al., 1999) Viscumin Viscum album 66 Type-II RIP Phase I (Eck et al., 1999; Tonevitsky et al., 1996)

ADP – Adenosine diphosphate; EF2 – Elongation factor 2; MAP – Microtubule-associated protein; Type-I RIP – N-glycosidase activity towards 28S rRNA; Type-II RIP – N-glycosidase activity towards 28S rRNA and cell surface binding domain. a – Molecular mass highly dependent of isoform (Akinrinmade et al., 2017). overlooked and may help to achieve this goal is the testing of different is internalized quickly, or rapidly recycled to the surface (Singh et al., RIT designs: a systematic analysis of benefitsand drawbacks when using 2020). Because all three characteristics are advantageous for immuno­ different mAbs, toxins and linkers. Therefore, we will discuss potential toxin efficacy on a cellular level but can be counter-productive on a sources and modificationsfor these three components as well as design tumor level, target antigen turnover and abundance on the cancer cell as principles in the next three sections. well as the affinity and avidity of the specificity-mediating domain should be balanced when designing a RIT. 6. Antibodies and epitopes for the targeting of cancer cells A natural size exclusion effect further contributes to tumor pene­ tration and the accumulation of immunotoxins (Shan et al., 2013). The design of RITs must accommodate effects at the cellular, tumor Whereas small-molecule drugs (<1.5 kDa) or small proteins (<40 kDa) and systemic levels. Key factors that act at the cellular level include can diffuse through the tumor tissue, resulting in homogenous but often target antigen abundance on the cancer cell surface, the internalization low intra-tumor accumulation (Golombek et al., 2018; Tang et al., rate of the target-RIT complex and the recycling rate of the target an­ 2014), therapeutic proteins in the size range 40~800 kDa (hydrody­ tigen to the surface. Whereas target abundance is also relevant for the namic radius 5~100 nm) show reduced transvascular diffusion, but therapeutic effect of conventional mAbs by triggering immunological once taken up tend to accumulate in tumors but not in healthy tissues (Li effector functions such as ADCC or complement-dependent cytotoxicity et al., 2017; Shan et al., 2013). Accordingly, full-size ADCs (~165 kDa) (CDC), rapid internalization is a key performance indicator for RITs and mAb-based RITs (~220 kDa, DAR = 2) penetrate tumors less effi­ because it determines the intracellular concentration of the toxic ciently than scFv-based counterparts (~60 kDa). payload, which may compete with effector functions (Peters and Brown, RIT size is also relevant on a systemic level because small scFv 2015). For example, despite a five-foldexcess of CD19 over CD22 on the immunotoxins are cleared more rapidly from the circulation, with a surface of lymphoma cells, a CD22-specific scFv RIT showed 140-fold serum half-life (t1/2) of only ~20 min compared to 4–8 h for full-size greater efficacy than an equivalent CD19-specific RIT because, even mAbs (Buhler et al., 2010; Shan et al., 2013). Because the specificity- though the Kd of each complex was similar (~7 nM), the CD22–RIT mediating domain of RITs is most flexible in terms of size (Fig. 1), it is complex was internalized five times faster (Du et al., 2008a). CD30, well suited to tune both tumor penetration and serum half-life. For CD64 and other surface markers have also been used to target hema­ example, the serum half-life doubled when a scFv RIT was converted to a tological cancer cells in various in vitro and in vivo studies (Mladenov bivalent version (Shan et al., 2013). Also, the Her2/neu-specific mAb et al., 2015; Palanca-Wessels and Press, 2014; Wayne et al., 2014) and Herceptin (trastuzumab) used in breast cancer therapy (Cao et al., 2014; the activity of immunotoxins typically increased with internalization Roses et al., 2009) was converted to a RIT by fusing either the full-size rate and the number of binding sites available (Kim et al., 2020; War­ antibody or its humanized scFv to the ~30 kDa recombinant RIP gelo­ galla and Reisfeld, 1989). nin from the Himalayan plant Gelonium multiflorum( Table 3) (Gilabert- Targeting solid tumors is more complex than the treatment of single- Oriol et al., 2017). The full-length Herceptin-Gelonin RIT was 1.3-fold cell hematological malignancies because tumors develop a binding-site more stable in circulation (t1/2 = 42 min) than the smaller scFv- barrier (BSB) that prevents RIT diffusion (Miao et al., 2016). BSBs Gelonin construct (t1/2 = 32.3 min), but the latter was superior in arise if the internalization of immunotoxins bound to the tumor surface terms of tumor penetration, probably due to the BSB on the tumor sur­ antigen is so rapid that the mass transport of molecules penetrating face (Cao et al., 2014). These results indicated that RIT size may be more deeper layers of the tumor is limited. This shielding or interception ef­ important than avidity, and that scFv RITs benefitfrom reduced BSB due fect is enhanced if the target antigen is overexpressed on the cell surface, to their size and single antigen binding site. However, the size and

8 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Fig. 3. Evolution of the toxins for the preparation of RITs using Pseudomonas aeruginosa exotoxin A (ETA) as a case study. The firstimmunotoxins joined the native ETA toxin (a) to full-size antibodies, including the signal peptide (yellow), receptor-binding domain I (green), translocation domain II (blue) and ADP ribosylation domain III (red). To prevent uptake into non-target cells, domain I was removed in the second generation of ETA-based immunotoxins (b). It is also possible to remove the translocation domain and introduce protease-sensitive linkers (magenta) (c) (Antignani and Fitzgerald, 2013). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) valency effects of RITs on therapeutic efficacyshould be studied in more indicates that VLS is a common side effect of many cancer therapeutics detail to provide insight into these competing effects. Biological modi­ and not RITs in particular. VLS can be ameliorated by mutating the fications such as glycosylation and/or the addition or deletion of do­ neonatal Fc receptor (FcRn) binding sites on mAbs, inhibiting the mains are preferred over chemical modifications such PEGylation nonspecificuptake of RITs into non-tumor cells, or by reducing the half- because they avoid additional process steps as discussed for ADC life of RITs in vivo to limit their contact time with the vasculature (Liu coupling. Modifying the specificity-mediating domain may also reduce et al., 2012). side effects caused by the toxic domain (see Section 7.1) Domain deletion can also remove antigenic sites from native toxins, Finally, it is important to remember when designing a RIT that the reducing their immunogenicity and increasing their serum half-life, ul­ specificity-mediating domain need not necessarily contain a mAb or timately allowing the repeated treatments necessary for certain diseases, derivative thereof because other proteins confer selective binding, such as the multiple doses of moxetumomab pasudotox required to treat including lectins and hormones. For example, Denileukin diftitox refractory hairy cell leukemia (Dhillon, 2018). Such antigenic sites are (Ontak) was the first FDA-approved therapeutic toxic fusion protein, often characterized by large hydrophilic amino acids and can be iden­ + indicated for the treatment of relapsed CD25 cutaneous T-cell lym­ tified experimentally by phage display technology or in silico based on phoma (CTCL) (Manoukian and Hagemeister, 2009). This consisted of the amino acid sequence or 3D structure using tools such as EpiMatrix diphtheria toxin linked to interleukin (IL)-2, and targeted cells positive (De Groot et al., 2008; Wei et al., 2018). Toxins may feature multiple for the IL-2 receptor CD25/IL2RA. Whether the term “immunotoxin” is immunogenic sites, as shown for the plant RIP trichosanthin, which is still appropriate for this molecule may be a matter of debate because IL-2 used in traditional Chinese medicine, and several alterations may is not an antibody but is nevertheless tightly linked with the immune therefore be necessary (Giansanti et al., 2018; Zhang et al., 2006). system. However, modifications to reduce immunogenicity should not cause unwanted secondary effects such as hyper-stability or loss of efficacy. 7. Toxins 7.2. Toxin uptake, mechanism of action and toxicity Proteinaceous toxins from bacteria, yeast, plants and humans have been used for the preparation of RITs (Table 3). Important RIT design Small-molecule drugs released from ADCs can easily diffuse through factors include the toxin structure and domains, the mechanism of ac­ the endosomal membrane into the cytosol, whereas protein toxins tion, intracellular translocation, the ease of cloning, expression and require active translocation mechanisms such as the endosomal acidi­ purification,and the potential for toxin immunogenicity (Antignani and fication of the diphtheria toxin or the KDEL-mediated retrograde traf­ Fitzgerald, 2013; Shapira and Benhar, 2010). ficking of ETA (Simon and FitzGerald, 2016). These mechanisms may involve partial or global unfolding of the RIT, as is the case for viscumin 7.1. Toxin structural features and their modification and ricin, respectively (Volynsky et al., 2019). For ricin, a five-helix structure in the middle of the toxicity-mediating A chain is sufficient The chemical inactivation of RIT toxin domains to prevent adverse for translocation to the cytosol in some cases (Vitetta et al., 1991). For effects is one option for modification, as successfully shown for several other toxins, features located in other domains may be necessary for immunotoxins containing ricin A, including Anti-MY9-bR targeting translocation, and this must be taken into account when modifying the + CD33 AML cells (Antignani and Fitzgerald, 2013; Choudhary et al., toxin (Lynch et al., 1997). After translocation, refolding in the cytosol 2011). However, the genetic engineering of modular toxins with sepa­ may therefore be necessary (Johnson et al., 1993; Johnson and Youle, rate domains for toxin activity, translocation and endocytosis is 1989), which often requires the presence of target-cell chaperones preferred because no additional process steps are required, as seen with (Antignani and Fitzgerald, 2013; Ratts et al., 2003). the three discrete domains of ETA (Fig. 3) (Weldon and Pastan, 2011). Toxins lacking the natural ability to cross membranes can be For example, removing nonspecific binding domains that promote the genetically fused to translocation domains such as ETA domain II uptake of full-length toxin RITs into non-target cells reduced the risk of (Mohammed et al., 2012). Alternatively, toxins such as cytolysin A and side effects such as vascular leak syndrome (VLS), thus favoring regu­ fragaceatoxin C can form a modular nanopore on the target cell surface latory approval (Alewine et al., 2015; Allahyari et al., 2017; Antignani without prior internalization (Azadpour et al., 2018; Mutter et al., and Fitzgerald, 2013). Other side effects, such as fluid retention in the 2018). This is advantageous because vesicle escape and refolding is stroma, peripheral edema, and cardiovascular failure due to increased rendered unnecessary and the corresponding RITs have shown IC50 vascular permeability, were not caused by the toxin but were triggered values in the range 246~2240 mg L-1 (4000~64,000 pM), but this must by IL-2-based therapeutics and those targeting cluster of differentiation be improved to compete with RITs based on other types of toxins. (CD) markers (Baluna and Vitetta, 1997; Jeong et al., 2019). This However, care must be taken to ensure that toxin mobility or

9 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683 internalization is only enhanced for target cells and not within the 8.1. Flexible linkers expression host, which could otherwise cause increased host cell damage. Flexible linkers promote efficientprotein folding because they allow The intracellular activity of most protein toxins involves one of three for rotation and bending of the peptide backbone, as shown for the key biochemical mechanisms, namely the ribosome inactivation by synthetically designed (Gly)8 linker (Sabourin et al., 2007). Flexibility is RIPs, inhibition of microtubule dynamics (Chen et al., 2017) or the conferred by small amino acid residues such as glycine because they lack formation of membrane pores (Escajadillo and Nizet, 2018; Walsh et al., a bulky side chain, but small polar side chains such as those of threonine 2013). Most RITs are based on toxins in the RIP category, probably and serine provide the additional benefit of forming hydrogen bonds because more than 100 different RIPs have been identified in bacteria with water molecules to improve solubility (Chen et al., 2013b). How­ and plants (Gilabert-Oriol et al., 2014) with similar structures and ever, domains connected via flexible linkers may unintentionally mechanisms of action (Walsh et al., 2013). Bacterial RIPs include ETA interact with each other via charged or hydrophobic surface patches, and diphtheria toxin, both of which inhibit protein synthesis by modi­ and this could block the activity or functionality of one or both domains, fying elongation factor 2 (Collier et al., 1979), whereas plant RIPs for example inhibiting the enzymatic mechanism of a toxin or covering include the lectins ricin and viscumin. Type I RIPs contain a single the antigen-binding site of an antibody (Amet et al., 2009; Chen et al., domain for N-glycosidase activity, whereas type II RIPs contain an 2013b). additional domain that mediates lectin-binding to the cell surface to promote endocytosis. As discussed above, the binding domain of toxins 8.2. Rigid linkers may be removed in the RIT design process. Additional yeast-derived RIPs have been incorporated into immu­ Rigid linkers keep the two domains apart and prevent unwanted notoxins, including the extracellular ribotoxins α-sarcin, RNase T1 and interactions. Such linkers may be composed of α-helices, such as the HtA, which target the highly conserved sarcin-ricin loop in ribosomal artificially designed helix-forming peptide linker sequence (EAAAK)n RNA, thereby blocking the translational machinery by inhibiting GTP which is stabilized by the electrostatic interactions between glutamic hydrolysis (Carreras-Sangra et al., 2008; Olombrada et al., 2017). Such acid and lysine side chains in each linker segment (Arai et al., 2001; ribotoxins have several advantages including their low natural immu­ Chen et al., 2013b). Non-helical rigid linkers can be stabilized by proline nogenicity, resistance to proteases, and thermostability (Carreras-San­ residues, often in the sequence (XP)n where X is any amino acid except gra et al., 2008). The latter is beneficialespecially for the production of proline and is preferably glutamine, lysine or alanine (Chen et al., RITs in whole plants if heat precipitation is used during downstream 2013b; George and Heringa, 2002). Such (XP)n stretches, typically ~14 processing to remove host cell proteins (Buyel et al., 2016; Menzel et al., amino acids in length, achieve a stiff and elongated conformation that 2016). The efficacy of ribotoxins has been demonstrated for the preserves the activity of fused protein domains (Evans et al., 1986) immunotoxin IMTXA33αs, which inhibited tumor growth and angio­ although additional residues (up to 34 in total) achieved the highest genesis in xenograft mouse models (Tom´e-Amat et al., 2015) but did not fusion protein activities in a comparative study (McCormick et al., induce T-cell activation as reported for a de-immunized variant (Jones 2001). et al., 2016; Olombrada et al., 2017). More recently, human cytolytic fusion proteins (hCFPs) have been developed as a novel class of RITs 8.3. Cleavable linkers comprising human (or humanized) antibody derivatives fused to human (and therefore non-immunogenic) cytolytic proteins such as The linker can also provide additional functions such as cysteine microtubule-associated protein tau (MAP tau), angiogenin, granzyme B residues to form intramolecular disulfide bridges or protease cleavage or death-associated protein kinase (DAPk) (Mungra et al., 2019). sites to facilitate intracellular toxin release. One example is the dithio­ Selecting a highly potent toxin can reduce the IC50 value of a RIT, cyclopeptide linker (LEAGCKNFF(PRS)FTSCGSLE) which contains an allowing lower doses and ultimately reducing costs. High potency can be intramolecular disulfide bond within its thrombin-sensitive PRS site achieved if the toxin has enzymatic activity. For example, the N-glyco­ (Nagamune, 2017). This linker undergoes rapid in vivo cleavage in the sidase activity of ricin can inactivate up to 2000 ribosomes per minute blood, with a serum half-life of <50 min, which is suitable for systemic (Endo and Tsurugi, 1988; Moshiri et al., 2016) resulting in a 30 μg kg-1 delivery but not for use in immunotoxins (Chen et al., 2010). Cleavable body mass lethal dose of ricin for 50% of the tested mice (LD50) when linkers in immunotoxins are generally based on target cell-specific applied by injection or inhalation (Audi et al., 2005). protease cleavage sites, allowing toxin release from the protease- activated prodrug after internalization or translocation (Vandooren 8. Linkers et al., 2016). For example, the efficacy of RITs has been increased 2–3- fold by using cleavable linkers that are sensitive to furin (Ruiz-de-la- Fusion proteins can be generated by joining the components directly, Herran´ et al., 2019; Wang et al., 2007), an endoprotease mainly local­ but the juxtaposition of two functional components that are not found ized in the trans-Golgi network and early endosomes (Thomas, 2002). together in nature may result in steric constraints that can cause protein Alternatively, cleavable linkers sensitive to cathepsin B, such as misfolding and ultimately low protein yields (Amet et al., 2009), the AGNRVRRSVG derived from the diphtheria toxin translocation domain accumulation of misfolded proteins (Zhao et al., 2008), the loss of (Yuan et al., 2011), can be used to release the toxin in the lysosome bioactivity (Bai and Shen, 2006), or unfavorable pharmacokinetic pro­ (Chen et al., 2013b; Yuan et al., 2011). files, for example due to fast immunotoxin clearance (Chen et al., 2013b). These issues can be addressed by inserting an intervening but 8.4. Linker stability in the host, during purification and delivery contiguous peptide linker. The choice of linker is important because the toxin component must remain stably attached to the antibody while in All linkers used for fusion protein expression in plants should be free the expression host, during purification and in the patients’ blood, but of plant-specificprotease cleavage sites to ensure structural integrity in must be released efficiently following uptake into target cells (Rühl the host cell. Therefore, linkers (and also the toxin and antibody com­ et al., 2018). The broad range of natural protein linkers that connect ponents) should be screened to identify and remove sites that are sen­ native multi-domain proteins can provide plenty of information about sitive to proteases expressed in the host plant (Hehle et al., 2016; preferable linker characteristics, including amino acid composition, Knodler¨ et al., 2019a; Zauner et al., 2018). Given the absence of a hydrophobicity and sequence length (Argos, 1990). So far, three major comprehensive database of plant proteases and their cleavage sites, the groups of linkers have been used in RITs: flexible, rigid, and cleavable. in silico prediction of cross-referenced cleavable amino acid sequences is not yet possible, and the suitability of different linkers may need to be

10 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683

Table 4 Properties of candidate linker sequences for immunotoxins in chronological order of publication.

Linker amino acid code Type Protease Protease compartment in tumor Reference Year sensitivity cell published

(EAAAK)n Rigid, None None (Arai et al., 2001; Takamatsu et al., 1990) 1990 α-helical (G4S)n Flexible None None (Brinkmann et al., 1993; van Rosmalen et al., 1993 2017) (TRHRQPRGWEQL) Cleavable Furin TGN, early endosome (Goyal and Batra, 2000) 2000 (GPLGMLSQ) Cleavable Gelatinase Matrix (Liu et al., 2000a) 2000 (AP)n Rigid None None (Mccormick et al., 2001 2001 (RAKR), (RKRR) Cleavable Furin TGN, early endosome (Zimmer et al., 2002) 2002 LEA(EAAAK)4ALEA Rigid, helical None None (Bai and Shen, 2006) 2006 (EAAAK)4A (AGNRVRRSVG) Cleavable Furin TGN, early endosome (Wang et al., 2007) 2007 (Gly)8 Flexible None None (Sabourin et al., 2007) 2007 (RHRQPRGWEQL) Cleavable Furin TGN, early endosome (Weldon et al., 2009) 2009 (LEAGCKNFF(PRS) Cleavable Thrombin Serum (Chen et al., 2010) 2010 FTSCGSLE) (GFLG)(R2KR6) Cleavable Cathepsin B, furin Lysosome, TGN, early endosome (Yuan et al., 2011) 2011 (R2KR6) Cleavable Furin TGN, early endosome (Yuan et al., 2011) 2011 (GFLG) Cleavable Cathepsin B Lysosome (Yuan et al., 2011) 2011 (FL) Cleavable Cathepsin B Lysosome (Shao et al., 2012) 2012 (RKKR) Cleavable Furin TGN, early endosome (Wein et al., 2012) 2012 (SGG)RHRQPRGWEQL(GGS) Cleavable Furin TGN, early endosome (Kaplan et al., 2016; Wei et al., 2018) 2016 (SGRSA), (SGKSA) Cleavable uPA Matrix (Braun et al., 2016; Liu et al., 2017) 2016

TEV – tobacco etch virus, TGN – trans-Golgi network, uPA – serine protease urokinase.

Fig. 4. Venn diagram of the different factors to consider for the production of recombinant immunotoxins (RITs) in plants: the antigen (not covered in this review), production platform (Section 5), antibody (Section 6), toxin (Section 7) and linker (Section 8). Each aspect has specific attributes that affect RIT activity and manufacturability. It is important that during RIT design these aspects are balanced and fine-tunedfor each specificapplication. DAR – drug-to-antibody ratio, cGMP – current good manufacturing practice; PTM – post-translational modification. determined empirically (Zischewski et al., 2016). High-throughput incubating them in the presence of plant extracts or mixtures of specific platforms such as tobacco plant cell packs are ideal for this because plant proteases. they can be screened in 96-well cultivation plates, allowing the testing of The optimal linker peptide for RITs should therefore stabilize the several thousand samples per week (Gengenbach et al., 2020; Rade­ fusion protein during synthesis, purification, storage and administra­ macher et al., 2019). The stability of candidate linkers could be tested by tion, but allow efficient in vivo cleavage by proteases present in the

11 M. Knodler¨ and J.F. Buyel Biotechnology Advances 47 (2021) 107683 endosomes or lysosomes of target cancer cells (Chen et al., 2010) in the framework of the Research Training Group “Tumor-targeted Drug (Table 4). Delivery” grant 331065168. This work was supported by the Fraunhofer-Gesellschaft Internal Programs under grant no. Attract 125- 9. Recommendations, conclusions and future perspectives 600164 and the state of North Rhine-Westphalia under the Leis­ tungszentrum grant no. 423 “Networked, adaptive production”. The five major aspects to consider when designing RITs are: the antigen (not discussed in this review), production platform (Sections 4 References and 5), antibody (Section 6), toxin (Section 7) and linker (Section 8) (Fig. 4). Antigen selection is important because it allows the therapeutic Akinrinmade, O.A., Jordaan, S., Hristodorov, D., Mladenov, R., Mungra, N., Chetty, S., index to be determined (effective dose in 50% of patients / lethal dose in Barth, S., 2017. 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