BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 17: Drug targeting Last time Intracellular drug de 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 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 on pulmonary, cardiovascular, and inflammatory diseases targeting to pathogen-infected cells od infected cells undergo changes in cell-surface molecule expression crossing blood-brain barrier Application Cellular target Molecular target Targeting ligand igand type Anti-cancer therapy Various tumor cells Folate receptor Folate Protein ligand for target EGF EGF receptor preferentially expressed on target Neovascular tissue B-FN(fibronectin anti-B-FN antibody antibody against fibronectin isoform only expressed during embryonic development nd in aggressive an tumors Anti-cancer Endothelial cells E-selectin receptor expressed at therapy, pulmonary P-selectin recepto sites of inflammation cardiovascular, and inflammatory 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 Anti-cancer therapy Transformed T IL-2Ra(interleukin-2 Anti-IL-2Ra antibody Antibody against target (T cell lymphomas) lymphocytes receptor a chain 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 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 targeting1 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 tumors2 o pulmonary, cardiovascular, and inflammatory diseases targeting to pathogen-infected cells o infected cells undergo changes in cell-surface molecule expression crossing blood-brain barrier3 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 LewisX 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- Targeted delivery receptor-Iigand-based targetin o/iigand pairs neral cell surface recept guide drug to target based on unique or over-expressed receptor on target cell type over-expressed in 95% of of non-mucinous ovan nian carcinomas high affinity (-1 nM Ko- typical half-life at 37C?) cons need to be humanized to avoid rapid opsonization on only variable region of mouse antibody need be retained for antigen recognition need to consider possible Fc receptor binding use of FAb fragments as an alternative source of ab 3d animation http://digilander.liberoit/danielefocosi/immunity.html#but,%20on%20the%20contrary%20of%20t CR,%20other%20BCR%20isotypes Key featuree of the protein atructure of (human lg G,)immunoglobulin molecules Fc receptor FAb/FAbO macrophage Lecture 17-Drug targeting 2 of 7
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Targeting Approaches4 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 carcinomas5 o antibody targeting pros • high affinity (~1 nM KD- 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 Fc receptor binding o Fc = ‘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. Fc Fc receptor macrophage FAb/FAbÕ -SH Lecture 17 – Drug targeting 2 of 7
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Antibody fragmentation enzymes Papain cleavag Pepsin Cleavage -SS-N -S.S. Flab)2 Fragment Fe Subfragments FAb framer Fe Subfragment Utility of antibody framer on Lack Fc region; reduced binding to phagocytic FcR-bearing macrophages and other phagocytes ou Reduced immunogenicity for non-humanized antibodies o FAb allows production of monovalent binding molecule Bivalent binding can trigger unwanted signaling cascades(e.g EGFR) a Unique chemical sites introduced at opened hinge region in FAb or F(Ab) Maleimide g- group MAL MAL MAL PEG surface Maleimide reaction with thiol creates stable thioether linkage onsoUrceofgraphichttp://www.nature.com/nrd/journal/v1/n7/slideshow/nrd838bx1.html Lecture 17-Drug targeting 3 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 HS SH Maleimide Eng-group B B + 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 C-N EDCNHS nDIT 分sH 5c-N~s⑥ (B-NH2Hydrazine -c-N⑥ &C-N-NHa 0(R-CHO i)NaBH3CN N^ SLinker-N⑧ Ns①}NHs Ns心Ma 3 N InkerS③ HOb NH-OH N-OH 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-1B, 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 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 sialyl lewis x receptor Therapeutic cargo IL-1B TNF Site of intibody-based targeting Targeted Activation local activation of a conjugate by action of enzymes or cellular environment example of cathepsin-sensitive linkages 'Reverse'targeting attraction of target cells to carrier Attraction of target cells to device via chemotaxis Dendritic ce∥s -1 per 100 cells in most tissues BSA rod BSA rod BSA rod MIP-3B/BSA rod+ BSA rod F BSA rod +OVA rodt b. MIP-3B/BSA rod+ OVA rod Tissue sections stained for MHc class ll(expressed by antigen-presenting cells) ( Kumamoto et al. 2002) Lecture 17-Drug targeting 5 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 lewisX 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’ targeting6 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 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 Objective: intracellular delivery of a cellular toxin to tumors o Target receptor also triggers receptor-mediated endocytosis (-) 680 incubation (min) (Shi et aL. 2002) Charge neutralization at low pH drives irreversible aggregation of particles at low pH(membrane fusion on particle aggregation) Rapid aggregation at 37C Lecture 17-Drug targeting 6 of 7
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. 20025 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 ③·⑧ 一￥￥ lanate Krcrptar m by FR-enpeod 助料(bmr Liposomes are electrostatically stabilized at neutral pH Change in net surface charge leads to membrane fusion within endosomes AraC cytosine-B-D-arabinofuranoside References Eniola, A O.& Hammer, D. A Artificial polymeric cells for targeted drug delivery. J Control Release 87, 15-22 2. Halin, C. et al. Enhancement of the antitumor activity of interleukin- 12 by targeted delivery to neovasculature. Nat Biotech/20,2649(2002) 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) 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) 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
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