ИСТИНА

Tobamoviruses are characterized by the capability for rapid and early synthesis of viral proteins, which ensures the rapid movement of viral genetic material through the plant and the accumulation of viral particles in quantities exceeding the yields of other plant viruses. The rapid synthesis of proteins and the rapid movement from cell to cell seem to allow tobamoviruses to outstrip the host in the development of protective reactions without requiring genes that encode silencing suppressors. This ability, along with the high stability of the virion, is one of the reasons that Tobacco mosaic virus (TMV) was the first virus discovered and led to the widespread use of TMV-based vectors in biotechnology. In recent years, vectors based on the genomes of tobamoviruses originally isolated from Brassicaceae species have become widespread in pharmaceuticals production using Nicotiana plants. The genome of Brassicaceae-infecting tobamoviruses has several characteristic features, with the most notable being MP/CP gene overlap. We believe that this peculiarity enables host range expansion and viral adaptation to Brassicaceae plants. We assume that maintaining the natural design of Brassicaceae-infecting tobamoviruses in the binary vector would enable the creation of an effective vector without significant modification of the original viral genome while including additional plant introns, which is a characteristic of magnICON vectors. Here, we have created an effective binary vector by considering the original natural genome organization of Brassicaceae-infecting tobamovirus. This vector provided effective synthesis of the heavy chain of trastuzumab and pertuzumab plant biosimilars (TPB and PPB, respectively) in Nicotiana benthamiana. We produced both biosimilars in wild-type (WT) N. benthamiana plant (PPB-WT and TPB-WT) using a Potato virus X-based vector and a new Brassicaceae-infecting tobamovirus-based vector. We assessed the efficiency of the tumour growth suppression induced by TPB-WT alone or in combination with PPB-WT. In accordance with our expectations, combined application of PPB-WT and TPB-WT increased the suppression of HER2/neu-positive tumour growth, confirming the functional activity of PPB-WT. Then, both biosimilars were produced in a transgenic XTFT N. benthamiana plant with knockout of the XT and FT genes (PPB-XTFT and TPB-XTFT). Our analysis confirmed the absence of 1,3-fucose and β1,2-xylose in the Asn297-linked glycan of PPB-XTFT and TPB-XTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that the PPB-WT and TPB-WT Asn297-linked glycans were mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297-linked GnGn-type glycans contained 1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguished these plant biosimilars from human IgG. Analysis of the TPB-XTFT total carbohydrate content indicated the possibility of changing the composition of the carbohydrate profile of not only the Fc but also the Fab portion of an antibody produced in transgenic XTFT N. benthamiana plants. Nevertheless, studying the antigen-binding capacity of the biosimilars showed that the absence of xylose and fucose residues in the Asn297-linked glycans did not affect the ability of the glycolmodified antibodies to interact with HER2/neu-positive cancer cells. Funding This work was supported by the Russian Science Foundation (project No. 16-14-00002).