A novel anti-CD22 scFv\u2013apoptin fusion protein induces apoptosis in malignant B-cells

Solmaz Agha Amiri,Mohammad Azizi,Mehryar Zargari,Soraya Shahhosseini,Najmeh Zarei,Dorsa Khorasanizadeh,Elahe Aminollahi,Vahid Khalaj,Faegheh Rezaie

doi:10.1186/s13568-017-0410-5

Abstract

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv–apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv–apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Highlights

Hematological malignancies are heterogeneous types of cancer and include a significant population of cancer patients
Expression analysis of the recombinant single-chain variable fragment (scFv)–apoptin The final expression cassette, pET–scFv–apoptin, was sequenced and the results confirmed that the scFv gene fragment was in V H to V L orientation and the (Gly4Ser)3 linker coding sequence was located between two chains
The presence of an immunoreactive band of ~44 kDa in western blot analysis of insoluble fraction confirmed the expressed protein (Fig. 2b). These findings revealed that the recombinant scFv–apoptin was successfully expressed mainly in insoluble form in E. coli BL21 (DE3)

Summary

Introduction

Hematological malignancies are heterogeneous types of cancer and include a significant population of cancer patients. B-cell malignancies are more common with an incidence rate of 21 per 100,000 (Keppler-Hafkemeyer et al 2000; Sant et al 2010; Weber et al 2015). Treatment outcomes in B-cell neoplasms have been greatly improved due to the availability of effective prognostic factors and the optimal use of conventional chemotherapeutic agents in combination with tumor specific monoclonal antibodies (mAbs) and stem cell transplantation (Hennessy et al 2004; Pui and Evans 2006). Some patients suffer from treatment refractory responses or even adverse effects related to the treatment (Coiffier et al 2002; Raut and Chakrabarti 2014). There is a need to develop novel and effective therapies with increased antitumor efficacy.

Methods

Results

Conclusion