Injectable hydrogels functionalized with antibody fragments as intra-articular cytokine sinks neutralizing pro-catabolic cytokines

Abstract

Purpose: Intra-articular (IA) injection with biologics like cytokine neutralizing antibodies to treat osteoarthritis (OA) have been explored, but so far success is limited. One of the key obstacle for the efficacy of intra-articular therapies is the short retention time of these biologics in the joint space due to rapid clearing by the synovial membrane. To overcome these limitations, we developed a new concept that potentially can increase the therapeutic value of pro-inflammatory cytokine neutralizing antibody fragments in the management of OA. The concept is based on the use of injectable hydrogels functionalized with the variable domain of single chain heavy chain only antibodies (VHHs) for cytokine capture. VHHs can be easily isolated using phage-display technology, recombinantly engineered and produced in yeast. These hydrogels can either be pre-crosslinked in microgels before injection or injected as polymer-VHH conjugates that cross link in a hydrogel in situ. Here, hyaluronic acid was used as model polymer after substitution with tyramine and maleimide groups. The tyramine moiety is used in an enzymatic cross-linking reaction to yield stable macromolecular hydrogel networks. The maleimide group is used for thiol-maleimide chemistry coupling a recombinantly engineered VHH with a free Cysteine in its C-terminal tail to the polymer backbone. We hypothesized that this strategy to functionalize hydrogels with the VHH can retain biological activity of neutralizing VHH. To proof the concept of these so-called cytokine sinks we have selected an anti-TNFα VHH that effectively neutralizes TNFα, which plays an essential role in cartilage degradation in inflammatory arthritis. Methods: To achieve directed conjugation of the VHH to the polymer, we have introduced an unpaired cysteine by using recombinant DNA technology in the C-terminus of the anti-TNFα VHH. Afterwards, the modified VHH was produced in yeast and purified. The conjugation of the VHH to the hyaluronic acid was performed by incubating the VHH with tyramine and maleimide functionalized hyaluronic acid. Characterization of binding affinity for both the non-conjugated VHH and conjugated VHH were measured by ELISA. hydrogels functionalized with the VHH was prepared by mixing the hyaluronic acid-VHH conjugates with enzyme peroxidase and H2O2. Biological activity of the conjugated VHH and hydrogels functionalized with the VHH was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα. An equimolar concentration of non-conjugated VHH was used as a control. Results: We show successful conjugation of the VHH to the polymer backbone. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. This modification did not affect the biological activity of the VHH. Using an NFκb responsive luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα and that this inhibition was comparable to inhibition by an equimolar concentration of non-conjugated VHH. Conclusions: We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for in situ generation of cytokine sinks capable of capturing different pro-catabolic cytokines in treatment of OA. Our Results demonstrate that cytokine sinks have great potential for neutralizing inflammatory pro-catabolic cytokines after intra-articular injection by increasing the retention time of neutralizing antibody fragments in the joint cavity.