Killer to cure: Expression and production costs calculation of tobacco plant‐made cancer‐immune checkpoint inhibitors

Abstract

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success.However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterised in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg-and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high-and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Plant‐derived Durvalumab variants show efficient PD‐1/PD‐L1 blockade and therapeutically favourable FcR binding

“…Plant expression systems have several advantages over conventional expression systems. The cultivation of plants under controlled conditions ensures the production of high‐quality protein in accordance with GMP (Good Manufacturing Practice) standards at lower costs (Buyel and Fischer, 2012 ; Diamos et al ., 2019 ; Ecker et al ., 2015 ; Ma et al ., 2017 ; Ridgley et al ., 2023 ). Moreover, the rapid growth, low cost of nutrient components and the avoidance of risks from contaminations with viruses and prions of animal origin are other arguments in favour of plant‐derived therapeutics.…”

“…So far PD‐L1 and PD‐1 antibodies produced in N. benthamiana plants were either decorated with plant‐specific glycan epitopes (core α1,3‐fucose and xylose), afucosylated or carrying high‐mannose glycans (Phakham et al ., 2021 ; Phetphoung et al ., 2022 ; Rattanapisit et al ., 2019 ; Ridgley et al ., 2023 ). The major drawbacks are that (i) mannosylation of therapeutic IgG antibodies increases their serum clearance by mannose receptors (Goetze et al ., 2011 ); (ii) classical plant CCD epitopes can lead to host antibody reactivity (Nkurunungi et al ., 2021 ); and (iii) lack of core fucose can significantly enhance antibody ADCC against PD‐L1‐positive non‐tumour cells (Yamane‐Ohnuki and Satoh, 2009 ).…”

“…Importantly, DL could be purified with yields up to 1 g/kg of fresh leaves, without the need for optimizing expression. Such recovery rates are significantly higher compared to previous reports on antibody accumulation levels (before purification) of 0.3 g/kg (Phakham et al ., 2021 ) and 0.7 g/kg upon co‐expression with a P19 silencing suppressor to optimize expression (Ridgley et al ., 2023 ).…”

“…A recent study generated a production cost model showing that the expression and purification recovery levels using plant platforms were as competitive as mammalian cell‐based platforms and therefore suitable to deliver accessible and more affordable diagnostic and therapeutic proteins (Ridgley et al ., 2023 ). Our research consolidates the scientific, clinical and economic potential of plant‐based expression platforms and dampens initial concerns relating to low yields and differences in glycosylation.…”

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco

“…Plants can have several advantages for the production of biopharmaceutical proteins such as low upstream production costs of ~ 50 € kg −1 wet biomass due to simple cultivation (Ridgley et al 2023 ) and an inherent safety because they do not support the replication of human pathogens (Schillberg et al 2019 ; Donini and Marusic 2019 ; Moustafa et al 2016 ; Tschofen et al 2016 ; Buyel 2023 ). However, the recovery of recombinant proteins from plant extracts can be challenging due to large quantities of particles and host cell proteins that are typically released during biomass homogenization (Wilken and Nikolov 2012 ; Buyel 2015 ).…”