Youngwook Won

Allosteric antibody-drug complex Current antibody-drug conjugate technology for targeted drug delivery is limited predominantly by the use of a chemical linker to conjugate anti-cancer agents to an antibody. To avoid the use of chemical conjugation, we have focused on biological affinity binding to incorporate anti-cancer agents to an antibody. This affinity binding is very simple and safe because this method does not require any chemical reactions that can alter the structure or activity of the anti-cancer agent or the antibody. Our linker-free technology allows eliminating the complicated steps necessary to remove organic solvents or excess chemicals. The goal of this project is to establish the feasibility of a biological affinity-driven antibody-drug complex as a targeted drug delivery platform that can leverage the advantages of antibody-drug conjugate technology, while overcoming its limitations. We have developed a bi-functional recombinant fusion protein containing a single-chain fragment variable (scFv) region and an anti-cancer drug-binding domain (ABD). In this scFv-ABD fusion protein, the ABD serves to capture its ligand anti-cancer agent through biological affinity binding that does not require the creation of chemical bonds, while the scFv offers a means of active targeting to the target cancer cells. The biological affinity-driven allosteric antibody-drug complex represents a new platform for targeted cancer drug delivery and therapy. Targeted chemoimmunotherapy Natural killer (NK) cells or T cells engineered by the virus-mediated gene transfer of chimeric antigen receptors (CAR)s are the most advanced approaches to selectively deliver active effector cells to eliminate specific cancer cells. The virus-mediated generation of CAR-NK or CAR-T cells, however, typically results in permanent CAR gene expression, making it very difficult to minimize the off-tumor toxicity of the CAR-NK or CAR-T cells. We have developed a NK cell-based chemoimmunotherapy that can take the advantages and overcome the limitations of current CAR technologies. This innovative method combines chemotherapy and immunotherapy through the embedding of antibody-drug conjugates (ADCs) on the surface of active NK cells. We hypothesize that these ADCs present on the surface of the NK cells can serve as a targeting moiety to specifically direct the active NK cells to the target tumor and deliver cytotoxic agents to destroy the target cancer cells. This strategy enables the targeted delivery of active NK cells as well as anti-cancer agents to the target tumor, thereby additively combining immunotherapy and chemotherapy to eradicate a specific tumor. Targeted stem cell delivery Mesenchymal stem cell (MSC) therapy for myocardial infarction (MI) has shown considerable promise in clinical trials. In order to achieve a clinical response, however, up to a billion MSCs need to be administered. This is because, with systemic infusion, only 1-2% of MSCs reach the ischemic myocardium. The reason for the low number of MSCs reaching the ischemic myocardium is the loss of the homing signal on the surface of the MSCs during their expansion in cell culture. Improving the homing of MSCs to ischemic myocardium offers the potential to improve the efficacy of MSC therapy. Stromal-derived factor-1 (SDF-1) is up regulated immediately after myocardial infarction and is released into the peripheral blood. SDF-1 reaches the bone marrow and recruits CXC chemokine receptor 4 (CXCR4)-positive stem cells. The CXCR4/SDF-1 axis plays an important role in MSC homing to ischemic myocardium. SDF-1 is highly expressed for only 48 hours after infarction while the therapeutic window to treat patients with acute MI (AMI) is only six hours. The current approaches to induce CXCR4 expression (i.e. genetic modification, hypoxic-preconditioning, and cytokine treatment) each require the culture of MSCs for 6 or more weeks, making them impractical for therapeutic clinical use. To allow the expression of CXCR4 within a clinically useful time frame, we have developed a novel method of MSC surface modification to incorporate recombinant CXCR4 protein (rCXCR4) on the cell membranes of allograft MSCs within 10 minutes. These cell surface modified MSCs migrate toward an SDF-1 gradient. Carrier-free gene delivery We have developed a Zinc/DNA cluster that can deliver plasmid DNA into cells without the need gene carriers. Despite the advantages of our method of carrier-free gene delivery over conventional polymeric gene carriers, the Zinc/DNA cluster does not deliver genes to specific target cells. Targeting ligands can be incorporated within the Zinc/DNA cluster, however, through Ligand-to-Metal Charge Transfer (LMCT). LMCT occurs between Zn2+ ions and the sulfhydryl group in cysteine. This interaction provides the fundamental chemistry necessary to incorporate targeting ligands within the Zinc/DNA cluster. The main goal of this project is to develop a platform to modify the Zinc/DNA cluster to allow for carrier-free and targeted gene delivery. LMCT between Zn2+ and cysteine (C) will be utilized to integrate either homing peptides (HPs) or recombinant antibodies (rAbs) within the Zinc/DNA cluster. Since most of the recently reported homing peptides have a common structure of C-XXXXXXX-C, there are two active sites to incorporate homing peptides into the Zinc/DNA cluster. Recombinant antibodies can be easily produced with an additional cysteine group at the C-terminus end, allowing for site-specific coordination of the recombinant antibody within the Zinc/DNA cluster.