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

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 characterized 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.

Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector.

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS-P system to investigate the expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti-HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct.

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors.

KEY MESSAGE: This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

[Cloning and expression analysis of CtbHLH transcription factor gene from Carthamus tinctorius and construction of plant expression vector].

To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.

A Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants.

Plant expression vectors are essential tools for gene functional analysis and molecular plant breeding. The gene of interest is transferred to the vector by molecular cloning technology. Nimble Cloning is a newly developed molecular cloning method with the advantages of simplicity, efficiency, and standardization. In this study, we developed a "pNC" vector system that contains 55 Nimble Cloning-compatible vectors for functional analysis of genes in plants. These vectors contain the NC frame flanked by unique adapters for one-step and standardized Nimble Cloning. We demonstrate that the pNC vectors are convenient and effective for the functional analysis of plant genes, including the study of gene ectopic expression, protein subcellular localization, protein-protein interaction, gene silencing (RNAi), virus-induced gene silencing, promoter activity, and CRISPR-Cas9-mediated genome editing. The "pNC" vector system represents a high-throughput toolkit that can facilitate the large-scale analysis of plant functional genomics.