Intra-articular injection with biologicals like pro-catabolic cytokine neutralizing antibodies to treat osteoarthritis (OA) is challenging due to the short retention time of these biologicals in the joint space. To address this issue, we first attach the antibody fragments to polymer conjugates that can form an hydrogel by in situ cross linking upon injection in the intra-articular space. To test this concept we have employed the variable domain of heavy chain of single chain only antibody (VHHs) neutralizing TNFα for cytokine capture. The aim of this study is to provide a proof of this strategy aimed at creating a cytokine sink in the joint space.
In this study, we have selected a VHH that effectively neutralizes TNFα. Recombinant DNA technology was used to introduce an unpaired cysteine in the C-terminus of the VHH. Hyaluronic acid was functionalized with maleimide, needed for coupling of VHH, and tyramine residues, needed for hydrogel formation. Hydrogels functionalized with the VHH were prepared by mixing the hyaluronic acid-VHH-tyramine conjugates with the enzyme peroxidase and H2O2. Biological activity of the functionalized hydrogels was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα.
We show successful conjugation of the VHH to the polymer backbone via thiol-maleimide chemistry, while maintaining the binding affinity of the VHH against TNFα. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. Using an NF-κB luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα.
We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for generation of cytokine sinks capable of capturing different pro-catabolic cytokines involved in OA after an intra-articular injection.