Targeted Reconstitution of Cytokine Activity upon Antigen Binding using Split Cytokine Antibody Fusion Proteins

Dario Venetz,Danil Koovely,Bruce Weder,Dario Neri

doi:10.1074/jbc.m116.737734

Abstract

The targeted assembly of antibody products upon antigen binding represents a novel strategy for the reconstitution of potent therapeutic activity at the site of disease, sparing healthy tissues. We demonstrate that interleukin-12, a heterodimeric pro-inflammatory cytokine consisting of the disulfide-linked p40 and p35 subunits, can be reconstituted by sequential reassembly of fusion proteins based on antibody fragments and interleukin-12 subunit mutants. Analysis of the immunostimulatory properties of interleukin-12 and its derivatives surprisingly revealed that the mutated p35 subunit partially retained the activity of the parental cytokine, whereas the p40 subunit alone was not able to stimulate T cells or natural killer cells. The concept of stepwise antibody-based reassembly of split cytokines could be useful for the development of other anticancer therapeutics with improved safety and tolerability.

Highlights

An ideal biopharmaceutical agent would display no toxicity when injected into the bloodstream, but should regain potent therapeutic activity upon binding to its antigen expressed on target cells
We have shown that fusion proteins, consisting of antibodies fused to mutants of the p40 and p35 subunits of mouse IL12, could selectively reassemble after product binding to the target antigen in vitro
Antibody-cytokine fusion proteins represent a promising class of biopharmaceutical agents [42], whose clinical potential is limited by toxicity associated with the cytokine payload

Summary

Introduction

We propose a new strategy for the development of antibody-cytokine fusions with activity on demand, based on the stepwise reassembly of cytokine subunits at the site of disease. Earlier studies on mouse IL12based antibody fusion proteins had revealed a preferential accumulation at the tumor site [32]. We have shown that fusion proteins, consisting of antibodies fused to mutants of the p40 and p35 subunits of mouse IL12, could selectively reassemble after product binding to the target antigen in vitro.

Results

Conclusion