Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

Abstract

Summary Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable antitumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 1 04-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia cofi (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma

“…Members of the pancreatic ribonuclease family have been proposed as possible alternatives to plant and bacterial toxins because these small extracellular proteins normally reside in the plasma and tissues of humans (Rybak and Newton 18 and references therein). The internalization of these proteins by microinjection, 19 antibodies, [20][21][22][23] or growth factors 24 has shown them to be potent cytotoxins in vitro, although significantly less so than antibody toxin constructs. For the first time, this paper describes a conjugate that is built with an RNA-damaging enzyme and differs significantly from immunotoxins with respect to mechanism of action and delivery.…”

A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I

“…Recombinant techniques were as described (Deonarain and Epenetos, 1998). Initial attempts to express DNase-I from the T7 expression system and E. coli BL21(DE3)/pLysS/E met with failure.…”

“…Cells were harvested and lysed as previously described (Deonarain and Epenetos, 1998;Deonarain et al, 1997), resuspending the bacteria in a buffer containing 50 mM Tris-HCl (pH 7.8), 2 mM CaCl 2 , 1 mM MgCl 2 . The soluble fraction was used to purify the soluble DNase-I by IMAC on nickel-Sepharose.…”

“…It was originally shown that the mammalian enzyme, bovine pancreatic ribonuclease (RNase A) could be as cytotoxic as ricin when micro-injected into Xenopus oocytes (Rybak et al, 1991). This observation led to the construction of a number of immunoconjugates based on angiogenin (Newton et al, 1996) and bovine seminal ribonuclease (Deonarain and Epenetos, 1998). These antibody-enzyme fusion proteins cause cell death by destroying cellular RNA.…”

“…The most important feature, however, for the use of this scFv in this study is that its antigen (hPLAP) has been implicated in the internalisation of IgGs upon ligand binding [its natural role is thought to be in the transport of maternal immunoglobulins to the foetus (Makiya and Stigbrand, 1992)], which makes it an attractive target for the delivery of a variety of agents. Its use in immunotoxins based on bovine seminal ribonuclease has been demonstrated (Deonarain and Epenetos, 1998).…”