Expression of anti-tumor recombinant IgG- and IgE-like genes in eukaryotic cells

Abstract

The tandem of humanized variable VL and VH genes (ScFv fragment 4D5) possessing a high affinity to the HER-2/neu oncogene (the epidermal growth factor receptor expressed in many types of human tumors) was attached through a flexible linker to the second exon of human antibodies of IgG or IgE isotypes constant gene. The humanized construct of IgE isotype was generated for the first time. Genes of the recombinant antibodies were cloned into the pCl-neo vector under the control of universal cytomegalovirus (CMV) promoter. Transfected HEK-293 cells efficiently produced antibodies of the corresponding isotypes IgE and IgG1. The results of Western blotting confirmed homogeneity of the expressed antibodies, which had the predicted molecular weight and specifically interacted with the HER-2/neu. The attachment of leader peptide to the 5'-end of the gene resulted in the preferential accumulation of recombinant antibodies in the cultural medium. These results indicate that de novo constructed humanized immunoglobulin genes express functionally active, singlechain recombinant antibodies in eukaryotic cells.

Suggestion the role of antibodies/immunoglobulins, produced by non-lymphoid cells, tissues and organs, as participants in different regulatory mechanisms

“…The results obtained also suggested a possibility for non-lymphoid cellular types, including neoplastic cells, to express genes for immunoglobulins, which is in agreement with the literature data [11][12][13]. It is possible this feature to be a step of the cascade regulatory pathways, in which molecules like GSH participate, and it has been additionally con rmed by the high levels of expression of immunoglobulins by non-lymphoid myeloblasts and other types of malignant cells [19][20][21].…”

“…Many ndings have explored the expression of genes coding domains of immunoglobulins/antibodies and membrane glycoprotein receptors in non-lymphoid cells [9,10], including by malignancies and malignant cells [11][12][13]. The performed investigations have also proved the possibility for production of membrane receptor glycoproteins by non-lymphoid and non-myeloid cells [14,15].…”