Multimerization through Pegylation Improves Pharmacokinetic Properties of scFv Fragments of GD2-Specific Antibodies.

Irina V Kholodenko,Sergey S Larin,Sergey M Deyev,Maria V Konovalova,Daniel V Kalinovsky,Alexey V Kibardin,Roman V Kholodenko,Tatyana V Shamanskaya,Elena V Svirshchevskaya,Igor I Doronin

doi:10.3390/molecules24213835

Abstract

Antigen-binding fragments of antibodies specific to the tumor-associated ganglioside GD2 are well poised to play a substantial role in modern GD2-targeted cancer therapies, however, rapid elimination from the body and reduced affinity compared to full-length antibodies limit their therapeutic potential. In this study, scFv fragments of GD2-specific antibodies 14.18 were produced in a mammalian expression system that specifically bind to ganglioside GD2, followed by site-directed pegylation to generate mono-, di-, and tetra-scFv fragments. Fractionated pegylated dimers and tetramers of scFv fragments showed significant increase of the binding to GD2 which was not accompanied by cross-reactivity with other gangliosides. Pegylated multimeric di-scFvs and tetra-scFvs exhibited cytotoxic effects in GD2-positive tumor cells, while their circulation time in blood significantly increased compared with monomeric antibody fragments. We also demonstrated a more efficient tumor uptake of the multimers in a syngeneic GD2-positive mouse cancer model. The findings of this study provide the rationale for improving therapeutic characteristics of GD2-specific antibody fragments by multimerization and propose a strategy to generate such molecules. On the basis of multimeric antibody fragments, bispecific antibodies and conjugates with cytotoxic drugs or radioactive isotopes may be developed that will possess improved pharmacokinetic and pharmacodynamic properties.

Highlights

Ganglioside GD2 represents one of the most attractive and promising targets for cancer immunotherapy
We show that the resulting scFv fragment constructs are capable of binding to the pure antigen adsorbed on an enzyme-linked immunosorbent assay (ELISA) plate, and to tumor cells expressing ganglioside GD2 on the cell surface
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Summary

Introduction

Ganglioside GD2 represents one of the most attractive and promising targets for cancer immunotherapy. This non-protein molecule belongs to the class of glycosphingolipids and is hyperexpressed primarily in tumors of neuroectodermal origin, whereas its expression on normal. Ganglioside GD2 is a marker of neuroblastoma, glioma, retinoblastoma, small cell lung cancer, melanoma, and various sarcomas [1,2]. One GD2-targeted immunotherapeutic drug, the chimeric monoclonal antibody dinutuximab (tradename Unituxin), is currently approved for clinical practice. It is used as part of combination therapy to treat high-risk neuroblastoma and increases the five-year patient survival by 20% compared to chemotherapy alone. Active research is currently being conducted regarding the choice of optimal combination of chemotherapies and Unituxin, as well as the selection of therapy regimens to enhance the efficiency of antitumor activity and reduce side effects in neuroblastoma patients [3]

Methods

Discussion

Conclusion