Development of therapeutic proteins based on a repeat protein scaffold

Abstract

Over the decades, chemotherapy has been mainly applied for treatment of various diseases. Despite of strong potency, chemical drug has often led to severe systemic toxicity in patients. The side effects are closely related with off-target effect of chemicals. To address the issue, immunoglobulin G (IgG) has been considered as an emerging class of therapeutics for targeted therapy due to its remarkable target specificity with high affinity. However, the therapeutic antibody has critical drawbacks as expensive production cost, poor cell penetration efficiency and low biophysical stability that are caused by complex expression and purification process, large molecular size and multimeric conformation. Hence, considerable effort has been made to develop the alternatives, and a variety of protein scaffolds have been reported including Affibody, DARPin, Nanobody and Repebody with significantly improved properties (stability, solubility and easy of engineering). Thus, non antibody protein therapeutics can hold great promise for being developed as the next generation of targeted therapeutics in treatment of various diseases. Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non-small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non-small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the tepebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6R$\alpha$ complex. Our results suggest that high affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non-small cell lung cancer. A targeted therapy using drug conjugates has attracted much attention, but efficient and site-specific drug conjugation methods have yet to be developed for enhance therapeutic efficacy. Here, we present a robust and site-selective approach to drug conjugation by enzymatic lipidation. Our approach comprises insertion of the CaaX sequence at the C-terminal end of a protein binder, prenylated using farnesyltransferase (FTase), followed by drug conjugation through oxime-forming reaction. We used monomethyl auristatin F (MMAF) and EGFR specific repebody (rEgH9) as an antitumor agent and protein binder, respectively. Our chemoenzymatic method enabled precisely controlled synthesis of drug conjugates with high yield (>95%) and a uniform drug-to repebody ratio (DRR) of 1.0 under mild conditions ($30^\circ C$, pH 7.4). The utility of our approach was demonstrated by showing a potent and selective cytotoxicity ($EC_{50}$ of 72 pM in HCC827 cells) and a remarkable anti-tumor activity of repebody-drug conjugates (RDCs) with negligible off-target effects in xenograft mice model. The present approach can be widely used for site-specific conjugation of a drug for a targeted therapy in a highly efficient and homogeneous manner. In this thesis, I have been successfully developed novel protein therapeutics using repebody engineering technology. The therapeutic protein candidates could expand the therapeutic market beyond the existing one and might result in creating new biological and medical industries. As mentioned above, the most important aspect is that as the RDCs are able to treat diseases which exhibit resistance or low potency against conventional therapies, it could make significant contributions to human health.