A single-chain variable fragment antibody against anti-leukemia agent, harringtonine as a tool for immunomodulation

Harringtonine (HT) is one of the cephalotaxus alkaloids firstly isolated from evergreen tree Cephalotaxus fortunei Hook f., which is distributed mainly in southern and northern China. Since HT has been shown to have antileukemic activity against mouse L-1210 leukemia and P-388 lymphocytic cells, it has been clinically studied and utilized for the treatment of the patient with acute leukemia and lymphoma [1]. However, the yield obtained from natural resources, genus Cephalotaxus, is very little; therefore, the studies to enhance production of HT and its downstream cephalotaxine have been one of the subjects of great interest in the field of medicinal plant breeding.

In our previous study, single-chain variable fragment (scFv) antibody was found to be powerful tool to enhance the production of secondary metabolites, when scFv genes were over-expressed in the host plants; the so-called immunomodulation [2]. As such, we are aiming at breeding Cephalotaxus species producing much amounts of HT.

In this study, we have constructed a scFv antibody against HT (HT-scFv), expressed by bacterial system, and characterized by enzyme-linked immunosorbent assay (ELISA) to use it as a tool for immunomodulation. The variable heavy and light chain genes were cloned directly from cDNA of hybridoma cell line 1D2, which secret highly-specific monoclonal antibody against HT, and assembled by splice overlap extension PCR using specific primers including flexible peptide (Gly₄Ser)₃ linker primers. The constructed scFv gene was ligated into the pET28a expression vector, expressed by E. coli BL21 (DE3). The denatured HT-scFv expressed as inclusion bodies in E. coli was solubilized and refolded by a stepwise dialysis method. The characterization of HT-scFv revealed that it retains highly-specificity to HT. In addition, its notable property enabled the development of an ELISA for the determination of HT. This study demonstrated the possibility of HT-scFv, which would be useful tool for both immunomodulation and quantitative analysis of HT.

Acknowledgements: The research in this paper was supported by Fukuoka Foundation for Sound Health Cancer Research Fund.

Keywords: immunomodulation, enzyme-linked immunosorbent assay, harringtonine, single-chain variable fragment (scFv) antibody.

References:

[1] Powell RG, Weislede D, Smith CR. Antitumor alkaloids from Cephalotaxus harringtonia: structure and activity. J Pharm Sci 1972; 61: 1227 – 1230

[2] Sakamoto S, Putalun W, Pongkitwitoon B, Juengwatanatrakul T, Shoyama Y, Tanaka H, Morimoto S. Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin. Plant Cell Rep 2012; 31: 103 – 110