Molecular farming for sustainable production of clinical-grade antimicrobial peptides

Abstract

Antimicrobial peptides (AMPs) are emerging as next-generation therapeutics due to their broad-spectrum activity against drug-resistant bacterial strains and their ability to eradicate biofilms, modulate immune responses, exert anti-inflammatory effects and improve disease management. They are produced through solid-phase peptide synthesis or in bacterial or yeast cells. Molecular farming, i.e. the production of biologics in plants, offers a low-cost, non-toxic, scalable and simple alternative platform to produce AMPs at a sustainable cost. In this review, we discuss the advantages of molecular farming for producing clinical-grade AMPs, advances in expression and purification systems and the cost advantage for industrial-scale production. We further review how 'green' production is filling the sustainability gap, streamlining patent and regulatory approvals and enabling successful clinical translations that demonstrate the future potential of AMPs produced by molecular farming. Finally, we discuss the regulatory challenges that need to be addressed to fully realize the potential of molecular farming-based AMP production for therapeutics.

Keywords: antimicrobial peptides; clinical‐grade AMP production; green synthesis; molecular farming.

Figure 1

Schematic representation of the mechanisms of antibiotic resistance and AMPs as promising approaches to combat resistant infections. Against the backdrop of increasing antimicrobial resistance, AMPs are emerging as an alternative antibiotic. The pharmacodynamics of AMPs are more favourable compared to the conventional antibiotics, and bacteria exhibit a lower propensity to develop resistance in comparison to antibiotics (Jochumsen et al., ; Kaufmann et al., ; Kintses et al., ; Rodriguez‐Rojas et al., 2014). Besides, AMPs show rapid killing kinetic (Fantner et al., 2010) through its stereotypical mechanism of creating pores in the cell wall, while other intracellular targets are also involved. Another important aspect of AMPs from a clinical perspective is their potential to be combined with existing antibiotics that have lost effectiveness due to resistance (Lazzaro et al., ; Lin et al., 2015), which raises the possibility of reviving antibiotics and achieving unprecedented potencies that have not been reported before. In context to produce AMPs for clinical application, SPPS standout as the primary method, and recombinant techniques can also be applied for large‐scale production.

Figure 2

Enhancing AMPs' clinical potential. (a) Overview of the failure of AMPs in clinical studies, including toxicity towards host cells, the rapid degradation of peptides in serum reducing their serum retention time and the high production cost associated with their manufacturing. (b) Possible strategies to improve the pharmacokinetic/pharmacodynamic properties of AMPs to achieve the same clinical outcomes as conventional antibiotics.

Figure 3

Harnessing the potential of the plant chassis: a comprehensive schematic of molecular farming methods to produce AMPs intended for clinical studies and the various stages involved in the approval process for plant‐produced peptide. EMA, European Medicines Agency.

Figure 4

Engineering the production of amidated peptides in plant cells. Agrobacterium tumefaciens is used to transfer DNA encoding AMP expression cassette to the nucleus of the transgenic plant cell expressing peptidylglycine α‐amidating mono‐oxygenase (PAM). Following this, glycine‐extended peptide mRNAs are transcribed as single or multiple transcripts, depending on the expression cassettes used. Geminiviral vector (BeYDV‐based) vectors undergo DNA amplification through rolling‐circle replication within the nucleus before transcription. Once transcripts leave the nucleus, they can be directly translated into proteins using pEAQ or BeYDV, or they can first undergo an amplification step (using, e.g., magnICON or TRBO) with the help of RNA‐dependent RNA polymerase (RdRP) and then be translated. Once translated, the glycine‐extended AMPs are amidated by PAM. For production of cyclic AMPs (cyclotides), AMPs are expressed as cyclotide precursors bearing an N‐terminal CXG motif and a C‐terminal DHV, which when they are co‐infiltrated with asparaginyl endopeptidases, leads to ligation of the two termini and cyclization of the peptides. PAM, peptidylglycine α‐amidating mono‐oxygenase; PHM, peptidylglycine α‐hydroxylating monooxygenase; PAL, peptidyl‐α‐hydroxyglycine α‐amidating lyase; TD, transmembrane domain; CD, cytoplasmic domain; ER, endoplasmic reticulum; BeYDV, bean yellow dwarf virus; pEAQ, ‘easy and quick’ plasmid vector; AEP, asparaginyl endopeptidases.

Figure 5

Plants as a chassis for sustainable biomanufacturing. Mammalian cell culture‐based processes relying on single‐use equipment and plant‐based processes both produce waste. Plant molecular farming typically yields a primary product comprising less than 1% of the total biomass produced in the initial stages of processing. However, the remaining biomass can be harnessed to produce additional value‐added products. These side streams have the potential to significantly enhance the economic value of the plant‐based process, potentially doubling revenue.