BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 17: Drug targeting Last time: Intracellular drug delivery Today: Drug targeting Reading: T.J. Wickham, ‘Ligand-directed targeting of genes to the site of disease,’ Nat. Med. 9(1) 135-139 (2003) Drug Targeting Applications of drug targeting 1 ? delivery of toxic drugs to tumors o highly toxic drugs that are too dangerous to deliver in a systemic manner ? e.g. potent radionuclides, cellular toxins ? allow smaller doses to be used ? delivery of DNA vectors to target cell type for genetic corrections ? targeting to vasculature o cancer treatment ? target to neovasculature forming around tumors 2 o pulmonary, cardiovascular, and inflammatory diseases ? targeting to pathogen-infected cells o infected cells undergo changes in cell-surface molecule expression ? crossing blood-brain barrier 3 Application Cellular target Molecular target Targeting ligand Ligand type Anti-cancer therapy Various tumor cells Folate receptor EGF receptor Folate EGF Protein ligand for target receptor preferentially expressed on target cells Neovascular tissue B-FN (fibronectin isoform) anti-B-FN antibody antibody against fibronectin isoform only expressed during embryonic development and in aggressive tumors Anti-cancer Endothelial cells E-selectin sialyl Lewis X receptor expressed at therapy, pulmonary, P-selectin receptor sites of inflammation cardiovascular, and inflammatory diseases Anti-cancer therapy Transformed B CD20 Anti-CD20 antibody Antibody against target (leukemias and B lymphocytes cell-surface protein cell lymphomas) unique to target class of cells (e.g. B cells) Anti-cancer therapy (T cell lymphomas) Transformed T lymphocytes IL-2Rα (interleukin-2 receptor a chain Anti-IL-2Rα antibody Antibody against target cell-surface protein not expressed on normal resting cells Lecture 17 – Drug targeting 1 of 7 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Targeting Approaches 4 Targeted delivery ? receptor-ligand-based targeting o general cell surface receptor-ligand pairs ? guide drug to target based on unique or over-expressed receptor on target cell type ? folate receptor ? over-expressed in 95% of non-mucinous ovarian carcinomas 5 o antibody targeting ? pros ? high affinity (~1 nM K D - typical half-life at 37°C?) ? high specificity ? cons ? need to be ‘humanized’ to avoid rapid opsonization o only variable region of mouse antibody need be retained for antigen recognition ? need to consider possible F c receptor binding o F c = ‘fragment crystallizable’ ? use of FAb fragments as an alternative ? source of Ab 3D animation: http://digilander.libero.it/danielefocosi/immunity.html#But,%20on%20the%20contrary%20of%20T cR,%20other%20BcR%20isotypes QuickTime? and a GIF decompressor are needed to see this picture. F c F c receptor macrophage FAb/FAb? -SH Lecture 17 – Drug targeting 2 of 7 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 FAb fragments Antibody fragmentation enzymes: papain papain Papain cleavage -S-S- (Pierce Chemical Co.) ? Utility of antibody fragments: o Lack Fc region; reduced binding to phagocytic FcR-bearing macrophages and other phagocytes o Reduced immunogenicity for non-humanized antibodies o FAb allows production of monovalent binding molecule ? Bivalent binding can trigger unwanted signaling cascades (e.g. EGFR) o Unique chemical sites introduced at opened hinge region in FAb’ or F(Ab’) 2 SHHS Maleimide Eng-group BB + Couple via QuickTime? and a Graphics decompressor are needed to see this picture. streptavidin to device -SH MAL PEG surface layer MAL MAL QuickTime? and a Graphics decompressor are needed to see this picture. ? Maleimide reaction with thiol creates stable thioether linkage: o Source of graphic: http://www.nature.com/nrd/journal/v1/n7/slideshow/nrd838_bx1.html Lecture 17 – Drug targeting 3 of 7 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 o Example: targeting to vasculature ? Inflammatory signals delivered from peripheral tissues to endothelial cells induce upregulation of ‘threat’ signals on the surface of these cells within the lumen of blood vessels o Cytokine signals such as IL-1β, TNF ? Chronic inflammation: upregulation of E-selectin ? Acute inflammation: upregulation of P-selectin o Used to direct neutrophils and monocytes to sites of inflammation Lecture 17 – Drug targeting 4 of 7 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 17 – Drug targeting 5 of 7 Site of inflammation IL-1β TNF sialyl lewis X receptor Therapeutic cargo ? antibody-based targeting Targeted Activation ? local activation of a conjugate by action of enzymes or cellular environment ? example of cathepsin-sensitive linkages ‘Reverse’ targeting 6 ? attraction of target cells to carrier Attraction of target cells to device via chemotaxis: (Kumamoto et al. 2002) Tissue sections stained for MHC class II (expressed by antigen-presenting cells) Dendritic cells ~1 per 100 cells in most tissues BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 17 – Drug targeting 6 of 7 Issues in Drug Targeting ? ‘collateral damage’ o how unique is target? Is it expressed in normal tissues ? often ratio of drug delivery tumor:normal tissue is not high enough ? In certain cases, elimination of healthy cells is acceptable ? E.g. hematopoietic system (T cells, B cells) can be replenished by bone marrow transplant ? Many times the normal tissue from which tumors are derived cannot be safely destroyed ? Tumor and viral escape o Loss of target antigen expression due to rapid mutations (antigen-loss variants) ? Immunological response to targeting agent o Early studies used mouse antibodies for targeting ? Low efficacy due to very short half-life and development of anti-sera Integrating targeting, activation, and intracellular delivery ? Example of targeted delivery to cytosol by functionalized pH-sensitive liposomal carriers o Shi et al. 2002 5 ? Objective: intracellular delivery of a cellular toxin to tumors o Target receptor also triggers receptor-mediated endocytosis (Shi et al. 2002) (+) (-) QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. ? Charge neutralization at low pH drives irreversible aggregation of particles at low pH (membrane fusion on particle aggregation) ? Rapid aggregation at 37°C BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 ? Liposomes are electrostatically stabilized at neutral pH ? Change in net surface charge leads to membrane fusion within endosomes ? AraC = cytosine-β-D-arabinofuranoside ? Cytotoxic agent for anti-tumor therapy References 1. Eniola, A. O. & Hammer, D. A. Artificial polymeric cells for targeted drug delivery. J Control Release 87, 15-22 (2003). 2. Halin, C. et al. Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature. Nat Biotechnol 20, 264-9 (2002). 3. Pardridge, W. M. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov 1, 131 9 (2002). 4. Wickham, T. J. Ligand-directed targeting of genes to the site of disease. Nat Med 9, 135-9 (2003). 5. Shi, G., Guo, W., Stephenson, S. M. & Lee, R. J. Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations. J Control Release 80, 309-19 (2002). 6. Kumamoto, T. et al. Induction of tumor-specific protective immunity by in situ Langerhans cell vaccine. Nat Biotechnol 20, 64-9 (2002). Lecture 17 – Drug targeting 7 of 7