Construction of an Integration Vector with a Chimeric Signal Peptide for the Expression of Monoclonal Antibodies in Mammalian Cells

Abstract

Antibodies are complex protein structures, and producing them using eukaryotic expression systems presents significant challenges. One frequently overlooked aspect of expression vectors is the nucleotide sequence encoding the signal peptide, which plays a pivotal role in facilitating the secretion of recombinant proteins. This study presents the development of an integrative vector, pVEAL3, for expressing full-length recombinant monoclonal antibodies in mammalian cells. The vector features a distinctive nucleotide sequence that encodes an artificial chimeric signal peptide with the following amino acid sequence: MMRTLILAVLLVYFCATVHC. Additionally, the vector incorporates several regulatory elements to enhance antibody expression, including the Gaussia luciferase signal sequence, internal ribosome entry site (IRES), P2A peptide, and a furin cleavage site. These elements coordinate to regulate the synthesis levels of the antibody chains. The analysis of clones obtained via transfection with the developed vector showed that over 95% of them secreted antibodies at levels significantly higher than those of the control. The immunochemical analysis of the chimeric antibody produced by the CHO-K1-10H10ch cell line confirmed the preservation of its functional activity.

Keywords: CHO-K1; artificial signal sequence; integrative vectors; recombinant antibodies.

Figure 1

Schematic diagram of the integration plasmid vector pVEAL3-10H10ch. 5′_SB and 3′_SB—transposase SB100X binding sites; CMV promoter—CMV promoter region; 176—nucleotide sequence encoding a hybrid signal peptide from luciferase (Cypridina noctiluca) and fibroin (Dendrolimus spectabilis), facilitating protein export from the cell; EMCV IRES—internal ribosome entry site; PuroR—nucleotide sequence encoding resistance to the antibiotic puromycin; SV40 poly (A) signal—nucleotide sequence stabilizing mRNA transcripts through polyadenylation; KanR—nucleotide sequence encoding resistance to the antibiotic kanamycin; ori—origin of replication. The complete nucleotide sequence of the developed vector is presented in Figure S1.

Figure 2

Schematic representation of the expression cassette. Key design elements are highlighted in color. Key elements of the construct: 176—nucleotide sequence encoding a hybrid signal peptide from luciferase (Cypridina noctiluca) and fibroin (Dendrolimus spectabilis), facilitating protein export from the cell; Furin—nucleotide sequence encoding the proteolytic site for the cellular protease furin; P2A—nucleotide sequence encoding the self-cleaving P2A peptide; GL—nucleotide sequence encoding the Gaussia luciferase signal sequence, facilitating protein export from the cell; EMCV IRES—internal ribosome entry site. Additional elements: CMV promoter—CMV promoter region; Puro—nucleotide sequence encoding the antibiotic resistance factor for puromycin.

Figure 3

Amino acid sequence of the chimeric signal peptide 176.

Figure 4

Optical density (OD) values of culture supernatant samples from 10H10ch antibody clones. The positive control, with a murine 10H10 antibody, had an OD of 1.4. The negative control, with a casein protein, had an OD of 0.04.

Figure 5

Electrophoresis in 15% SDS-PAGE. Lane 1—recombinant 10H10ch antibody under denaturing conditions (concentration ~10 µg/well); lane 2—murine 10H10 antibody under denaturing conditions (concentration ~20 µg/well); lane 3—molecular weight markers (250–10 kDa). The recombinant 10H10ch antibody was isolated using affinity chromatography. The murine 10H10 was isolated using a caprylic acid purification protocol from mouse ascites fluid.

Figure 6

The results of the interaction of the recombinant 10H10ch and murine 10H10 antibodies with recombinant fragments of flavivirus envelope proteins are as follows: TEF1 represents the 1 + 2 domains of the E protein from tick-borne encephalitis virus; ZEF1 denotes the 1 + 2 domains of the E protein from Zika virus; WEF1 refers to the 1 + 2 domains of the E protein from West Nile virus; DEF1 comprises the 1 + 2 domains of the E protein from Dengue virus; and TNS1 indicates the non-structural protein 1 from tick-borne encephalitis virus.