Disease-triggered release of a TNFA neutralizing VHH for intra-articular disease modification

Purpose (the aim of the study): The aim of this project is the development of an intra-articular injectable drug depot for disease triggered release of a tumor necrosis factor α (TNFα) neutralizing variable domain of single chain heavy chain only antibody (VHH). The use of a VHH as therapeutic has several advantages over conventional antibodies for disease modification in degenerative joint diseases like osteoarthritis and rheumatoid arthritis. VHH are e.g. much smaller, can be easy and cheaply produced in yeast and are more resistant to chemical modifications. However, due to their small size VHHs are rapidly cleared from the joint after an intra-articular injection. Here we describe a mechanism to increase the half-life of the VHH in the joint by conjugating the VHH to tyramine modified hyaluronic acid which crosslinks in vivo into a stable hydrogel upon mixing with the enzyme horseradish peroxidase (HRP) and minute amounts of hydrogen peroxide (H2O2) during intra-articular injection. The VHHs are released from this network by enzymatic degradation of the hydrogel mediated by the enzyme hyaluronidase.

Methods: A TNFα-targeting VHH was selected using llama immunization and phage-display technique. The neutralizing activity of the VHH was tested in vitro using a HEK293 NF-kB reporter cell line and was compared to the commercial antibody Adalimumab. A free cysteine residue was introduced in the C-terminal tail of the VHH by genetic engineering. Hyaluronic acid (HA, mw 27 kDa) was functionalized with tyramine (TA) and maleimide (Mal) groups and analysed with 1H-NMR. Using the engineered free cysteine introduced in the C-terminus, VHH were covalently coupled to the Mal groups of the polymers. The binding affinity of the VHH after coupling was evaluated using ELISA and surface plasmon resonance imaging (SPRi). The VHH-polymer conjugates were mixed with HRP and H2O2 for almost instantaneous hydrogel formation via induction of tyramine-tyramine crosslinks. VHH release from the gels was tested by incubation with hyaluronidase and the biological activity of the released VHHs was measured by ELISA over a period of four weeks. Lastly, the VHH-containing hydrogels were tested in vitro with a trans-well assay and ex vivo using cartilage explants for TNFα neutralizing activity.

Results: The VHH showed complete inhibition of TNFα stimulation when administered at a 100 times molar excess, where Adalimumab only inhibited 30% at the same molar excess. HA was successfully functionalized with TA and Mal groups. 1H-NMR spectra of the polymers showed that the number of substituted TA and Mal moieties per 100 disaccharide units was 7.5 and 12 in HA-TA-Mal, respectively. Directed coupling of the VHH via the engineered free cysteine to the maleimide group in HA was highly successful with a coupling efficiency of 95%. The binding affinity of the VHH was minimally affected by the conjugation process. Upon mixing of the VHH-conjugated polymers with the HRP and H2O2 stable hydrogels were formed. These VHH-functionalized hydrogels efficiently neutralized TNFα activity in a reporter cell line and in an ex vivo cartilage explant culture model. Incubating the VHH-functionalized hydrogels with hyaluronidase resulted in gradual hydrogel degradation and thus release of the VHH. A small burst release of VHH was observed during the first few days after hydrogel formation due to the diffusion mediated release of non-conjugated VHHs which was followed by a first order release over a period of several weeks. The released VHHs remained biologically active.

Conclusions: We have developed an injectable in situ gelating hydrogel modified with a TNFα neutralizing VHH. The release of the VHHs is controlled by hyaluronidase-mediated hydrogel degradation. Since hyaluronidase activity is upregulated in a variety of joint diseases, hydrogel degradation and thus release of the disease modifying VHH is controlled by disease activity. This platform technology can be used as a universal system for development of long lasting intra-articular drug depots of which the release of the active compound is tightly controlled by locally produced enzymes of which the activity depends on disease severity. Such platforms may be used to locally disrupt the vicious cycle of inflammation and joint degradation.