Sungkwon Kang

Self-assembled nanoplatform for targeted delivery of chemotherapy agents via affinity-regulated molecular interactions.

Site-specific delivery of drugs while minimizing unwanted distribution has been one of the pursued goals in cancer therapy. In this endeavor, we have developed targeted polymeric nanoparticles called amphiphilic urethane acrylate nonionomer (UAN) for encapsulation of diverse water-insoluble drugs and diagnostic agents, as well as for simple and reproducible surface conjugation of targeting ligands. Using monoclonal antibodies or lymphocyte function-associated antigen-1 (LFA-1) I domain engineered for varying affinities to intercellular adhesion molecule (ICAM)-1, we were able to deliver UAN nanoparticles to human cancer cells with the efficiency dependent on the strength of the molecular interactions and the degree of ICAM-1 expression on cell surface. Compared to non-specific uptake of free drugs, targeted delivery of UAN nanoparticles carrying equal amount of drugs produced more potent cytotoxicity. Notably, without the targeting ligands attached, UAN nanoparticles were largely precluded from non-specific uptake by the cells, resulting in much lower toxicity. The versatility of our UAN nanoparticles in both payload encapsulation and presentation of targeting ligands may facilitate developing a robust platform for evaluating various combinations of cancer drugs and molecular interactions toward developing effective cancer therapy formulations.

Tumor suppression via paclitaxel-loaded drug carriers that target inflammation marker upregulated in tumor vasculature and macrophages

Clinically approved chemotherapeutic nanoparticles may provide advantages over free drugs by achieving slower clearance and preferential accumulation in tumors. However, the lack of leaky vasculatures can create barriers to the permeation of ~100 nm-sized nanoparticles in solid tumors. We hypothesized that nanoparticles designed to target both tumor and tumor stroma would penetrate deeper into the tumors. To construct such comprehensive drug carriers, we utilized cross-linked amphiphilic polymer nanoparticles and functionalized them to target ICAM-1, a biomarker prevalent in various tumors and inflamed tumor stroma. The targeting moiety was derived from the modular domain present in α(L) integrin, which was engineered for high affinity and cross-reactivity with human and murine ICAM-1. ICAM-1-selective delivery of paclitaxel produced potent tumor suppression of not only ICAM-1-positive cervical cancer cells but also ICAM-1-negative tumors, presumably by causing cytotoxicity in tumor-associated endothelium (CD31(+)) and macrophages (CD68(+)) over-expressing ICAM-1. Contrary to the strategies of targeting only the tumor or specific tumor stromal constituents, we present a strategy in delivering therapeutics to the major cellular components of solid tumors. Drug carriers against inflammation-biomarkers may be effective against many different types of tumors, while being less susceptible to the highly mutable nature of tumor markers.

Tunable physiologic interactions of adhesion molecules for inflamed cell-selective drug delivery

Dysregulated inflammation contributes to the pathogenesis of various diseases. Therapeutic efficacy of anti-inflammatory agents, however, falls short against resilient inflammatory responses, whereas long-term and high-dose systemic administration can cause adverse side effects. Site-directed drug delivery systems would thus render more effective and safer treatments by increasing local dosage and minimizing toxicity. Nonetheless, achieving clinically effective targeted delivery to inflammatory sites has been difficult due to diverse cellular players involved in immunity and endogenous targets being expressed at basal levels. Here we exploit a physiological molecular interaction between intercellular adhesion molecule (ICAM)-1 and lymphocyte function associated antigen (LFA)-1 to deliver a potent anti-inflammatory drug, celastrol, specifically and comprehensively to inflamed cells. We found that affinity and avidity adjusted inserted (I) domain, the major binding site of LFA-1, on liposome surface enhanced the specificity toward lipopolysaccharides (LPS)-treated or inflamed endothelial cells (HMEC-1) and monocytes (THP-1) via ICAM-1 overexpression, reflecting inherent affinity and avidity modulation of these molecules in physiology. Targeted delivery of celastrol protected cells from recurring LPS challenges, suppressing pro-inflammatory responses and inflammation-induced cell proliferation. Targeted delivery also blocked THP-1 adhesion to inflamed HMEC-1, forming barriers to immune cell accumulation and to aggravating inflammatory signals. Our results demonstrate affinity and avidity of targeting moieties on nanoparticles as important design parameters to ensure specificity and avoid toxicities. We anticipate that such tunable physiologic interactions could be used for designing effective drug carriers for in vivo applications and contribute to treating a range of immune and inflammatory diseases.

Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies

A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (αMβ2) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigen–antibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications.

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

A novel in vitro ischemia/reperfusion injury model

The reperfusion of blood flow occurred in a number of conditions such as stroke and organ transplantation immensely augments tissue injury and causes more severe damage than prolonged ischemia. In the present study, we designed a novel double-layer parallel-plate flow chamber (PPFC) to develop an in vitro ischemia/reperfusion (I/R) injury model and examined the effects of I/R on inflammatory responses in human microvascular endothelial cells (HMEC-1). The expression of pro-inflammatory mediators, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) in HMEC-1 was measured by quantitative real-time RT-PCR. The cells were also pre-treated with antioxidant pyrrolidine dithiocarbamate (PDTC) to verify involvement of an oxidative mechanism in I/R injury in vitro. The morphological changes and attenuated expression of pro-inflammatory mediators were observed in HMCE-1 exposed to the physiological flow. In contrast, I/R markedly and significantly up-regulated expression of pro-inflammatory mediators in HMEC-1. Additionally, pretreatment with PDTC significantly reduced I/R-mediated overexpression of pro-inflammatory mediators. The data from the present study provide evidence demonstrating that our newly designed PPFC can be utilized as an effective in vitro cell culture model system to develop new drugs specifically targeting against ischemia/reperfusion (I/R) injury.

Complex structure of engineered modular domains defining molecular interaction between ICAM-1 and integrin LFA-1.

Intermolecular contacts between integrin LFA-1 (αLβ2) and ICAM-1 derive solely from the integrin αL I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the αL I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine αL I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the αL I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to αL I domain but reduced interaction with HRV.

A novel double-layer parallel-plate flow chamber

In the present study, we designed and developed a novel double-layer parallel-plate flow chamber (PPFC) to study the dynamic response of vascular endothelial cells to controlled levels of shear stress. In addition, the effects of fluid shear stress on the structure and function of endothelial cells were examined to validate whether a novel PPFC can be employed for studies in the areas of biomedical research. Human microvascular endothelial cells (HMEC-1) were either maintained in static condition or exposed to laminar flow for 24 h. The morphological changes and attenuated expression of pro-inflammatory mediators, such as ICAM-1, VCAM-1, and E-selectin were observed in endothelial cells exposed to the flow. HMEC-1 cells were also either maintained in continuous laminar flow (Normal flow) or subjected to 1 h of flow cessation followed by reperfusion (12 h) of flow (Ischemia/Reperfusion). The real-time RT-PCR analysis showed that Ischemia/Reperfusion significantly up-regulated expression of pro-inflammatory mediators in HMEC-1 compared to Normal flow, which is consistent with previous in vivo studies. These data indicate that our newly designed PPFC can provide a better in vitro system for versatile applications of biomedical research.