Enzyme responsive delivery of engineered antibody fragments
Lin Zhong is a PhD student in the department Developmental BioEngineering. (Co)Promotors are prof.dr. H.B.J. Karperien and dr. B. Zoetebier from the faculty of Science & Technology.
Therapeutic proteins including antibodies (or antibody fragments), cytokines and growth factors are a rapidly expanding drug class in clinical use for treatment of various diseases. However, in vivo protein delivery remains a challenge because of their instability and short half-life, often resulting in poor drug bioavailability at disease sites. Hydrogels are extensively studied for the localized and sustained delivery of proteins due to their excellent biocompatibility, tunable biodegradability and ability to mimic native extracellular matrix (ECM). For instance, injectable hydrogels as drug delivery system (DDS) can be applied to deliver these therapeutic proteins and to increase their retention time in the joint space resulting in enhanced osteoarthritis (OA) treatment effects. To improve the release kinetics of proteins from hydrogels the complementary use of both chemical and genetic methods, by engineering proteins with intrinsic features such as a proteinase cleavage site, reactive handle for bioconjugation, and affinity domain for interaction with materials, may provide a new concept in designing a controlled release system for protein delivery.
This thesis describes two different hydrogel-based systems for controlled delivery of the variable domain of the heavy chain only antibodies (VHHs) in response to environmental cues such as hyaluronidase and matrix metalloproteinases (MMPs). Covalent conjugation was applied to immobilize the engineered VHHs on the polymer backbone within a hydrogel, either through stable or cleavable linkers. To achieve this, we explored in chapter 2 conjugation strategies for introducing maleimide (Mal) and tyramine (TA) functional groups onto the backbone of the natural polymer dextran. The tyramine groups ensure rapid in situ crosslinking of the polymers into hydrogels upon co-injection of the conjugates involving horseradish peroxidase (HRP) as a catalyst with hydrogen peroxide (H2O2) as an oxidant. The maleimide group can be used for the directed coupling of the biomolecules such as VHHs genetically engineered to express an unpaired free cysteine in their C-terminal tail using thiol-maleimide chemistry.
In chapter 3, we established an efficient protocol for the conjugation of a genetically engineered VHH to dextran and hyaluronic acid using thiol-maleimide chemistry. We showed that conjugation efficiency is dependent on the identity of the used VHH. Most likely the conjugation efficiency was dependent on the accessibility of the unpaired free cysteine in the VHH. We showed that conjugation efficiency can be increased by increasing the accessibility of the unpaired cysteine by incorporating a flexible Glycine/Serine linker between the complementary determining region of the VHH and the unpaired cysteine, or by extending the C-terminal tail by incorporating a peptide sequence that could be cleaved by a matrix metalloprotease or by introducing more than one free cysteine in the C-terminal tail. We furthermore showed that the conjugation marginally impacted the antigen binding properties of the VHH.
In chapter 4, we have used this strategy to generate conjugates of hyaluronic acid tyramine and a Tumor Necrosis Factor α (TNFα) neutralizing VHH. Upon mixing with HRP and H2O2 injectable hydrogels were obtained. These hydrogels efficiently captured TNFα and neutralized their activity in bioassays using both in vitro cell models and ex vivo explant cultures. We furthermore showed that exposure of the hydrogel to the enzyme hyaluronidase resulted in the controlled release of the VHH into the medium due to network degradation in a concentration dependent manner. This system could be used as an intra-articular injectable hydrogel neutralizing pro-inflammatory cytokines in a diseased joint.
In chapter 5, we report on the development of a novel hydrogel system based on the conjugation of a VHH genetically engineered to express a Matrix Metalloproteinase 13 (MMP13) cleavable peptide sequence between the free cysteine in the C-terminal tail and the Complementarity Determining Region 3 (CDR3 region) of the VHH to a dextran-TA-Mal backbone using thiol-maleimide chemistry. The dextran-TA / VHH conjugate could be formulated into monodisperse microgels and we showed the subsequent release of the VHH after treatment of the microgels with activated MMP13. Since MMP13 is one of the enzymes involved in cartilage breakdown during osteoarthritis, this work opens up the possibility to develop an injectable drug depot of which the release of a potentially disease modifying osteoarthritic VHH from the microgels is controlled by the disease state.
In chapter 6, we have performed a proof of concept study in a rat model to show that conjugation of a VHH to dextran or hyaluronic acid tyramine conjugates increases the retention time of the VHH in the joint. Indeed, we showed the presence of VHH in the joint up to 3 months using fluorescent imaging and immunohistochemistry at the end of the study. Contrary to expectation, a near infrared-labelled bare VHH was also still detectable in the joint 3 months after injection based on non-invasive longitudinal fluorescent imaging. Nevertheless, the experiment showed a proof of concept that conjugation of a VHH to a polymer backbone can retain the VHH in the joint at the place of injection.
Finally, in chapter 7, a general discussion is provided summarizing some of the possibilities and limitations of these studies. We conclude that the two hydrogel-based delivery systems described in this thesis and which involve genetic engineering of VHHs and covalent conjugation of VHHs to polymer backbones within hydrogels, provide innovative strategies to enable VHH release in a controlled way, with release kinetics governed primarily by the degradation properties of the HA hydrogel or cleavage rate of linkages between VHHs and polymers in response to local enzyme activity in the OA joint. More broadly, this thesis offers a new paradigm in design of stimuli-responsive delivery systems of biologicals for a prolonged period of time based on pathological or the natural physiological features of the tissue, opening up new possibilities for the delivery of various therapeutic proteins.
