The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

Abstract

Background Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. Results The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. Conclusion The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

The lyophilized chloroplasts store synthetic DARPin G3 as bioactive encapsulated organelles

“…We recently reported the high-level expression of DARPin G3 (about 3.4 mg/g leaf fresh weight) in the chloroplast of tobacco plants as the first antibody mimetic produced in a plant expression system. Its functionality in HER2 imaging is comparable to that of previously produced DARPin G3 in E. coli and P. pastoris [ 19 ]. In this study, we investigate the long-term storage effect (for nine months at 22–24 °C) on the stability and functionality of chloroplast-made DARPin G3 in lyophilized leaves and isolated chloroplasts of transplastomic tobacco plants.…”

“…Transplastomic plants could thus lead to a significant breakthrough in biotechnology, ranging from medication development to the large-scale commercial synthesis of enzymes and biofuels. As summarized in several reviews, chloroplast transformation is expected to offer unique advantages in the advancement of molecular farming for the production of biopharmaceuticals, vaccines, antibiotics, anti-microbial peptides, phytoremediation, industrial enzymes, biofuels, biomaterials [ 28 , 29 ], and more recently, scaffold proteins as antibody mimetics [ 19 ]. Several functional therapeutic proteins have been expressed in tobacco chloroplasts in quantities comparable to or greater than those found in animal or bacterial bioreactors [ 30 , 31 ].…”

“…We recently and for the first time developed tobacco transplastomic lines producing high levels of DARPin G3 as an antibody mimetic for HER2 targeting in HER2-positive cancers [ 19 ]. Our previous study’s results demonstrated that the DARPin G3 produced in chloroplasts binds HER2 with high affinity, as previously reported for this DARPin G3 expressed in E. coli [ 20 ] or P. pastoris [ 17 ], as shown using an enzyme-linked immunosorbent assay (ELISA).…”

Designing and computational analyzing of chimeric long-lasting GLP-1 receptor agonists for type 2 diabetes

“…In incubation with human serum (ex vivo), no proteolytic digestion was observed, indicating high stability for potential drug use. E. coli , yeast, and plants can all express DARPins at high levels in the soluble form 31 , 33 , 34 , and based on chemical characterization, DARPins are predicted to be non-immunogenic 51 . The albumin-binding DARPin domain demonstrated nanomolar affinities for humans, cynomolgus monkeys, mice, and rats’ serum albumin at pH 7.4 or 6.0.…”

“…Designed ankyrin repeat proteins are a promising class of small non-immunoglobulin binding proteins that bind target proteins with high affinity and specificity 29 , 30 . DARPin domains have desirable biophysical features, such as high thermal and thermodynamic stability, and can be expressed in high quantities in yeast, bacteria, and tobacco chloroplast expression systems 31 – 33 . Serum albumin-binding DARPin domains can be used to extend the terminal half-lives of next-generation protein therapeutics 28 .…”