Assessment of selective toxicity of insect cell expressed recombinant A1-GMCSF protein toward GMCSF receptor bearing tumor cells

Abstract

One of the emerging therapeutic strategies for targeted treatment of most cancers is the use of immunotoxins which are fusion proteins consisted of a targeting and a toxic moieties. We previously showed that the recombinant A254-GMCSF fusion protein selectively kills acute myeloblastic leukemia cells which harbor a large number of granulocyte-macrophage colony stimulating factor (GMCSF) receptors. Since further in vitro and preclinical studies require large amounts of this fusion protein free from any troublesome material like lipopolysacharide, we selected the insect cell expression system. Thus, the coding sequences of the A254-GMCSF and its truncated form, A247-GMCSF, were cloned and expressed by Sf9 cells. Subsequently, specific cytotoxicity of the purified proteins was evaluated on GMCSF receptor positive cell lines. SDS-PAGE and Western blot analysis of the expressed A254GMCSF and A247GMCSF fragments revealed bands of about 60 kD which were larger than the theoretically predicted size of about 47 kD. Deglycosylation analysis showed that these proteins are N-glycosylated by the insect cells. However, any other post-translation modification of the proteins by insect cells could be the reason for higher molecular weight of the fragments. Cytotoxicity assays showed specific killing activity of these proteins on HL60 and U937 cell lines with IC₅₀s ranging 2-2.5 µg/ml. These IC₅₀ values are much higher than those obtained from bacterially expressed A254-GMCSF (80 ng/ml) which could be due to any modification performed by insect cells on the fusion proteins.