Lecture 18

Lecture 18 - BioE10 Lecture 18 Professor Irina Conboy...

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Unformatted text preview: BioE10: Lecture 18 Professor Irina Conboy BioE10: Lecture 18 Professor Irina Conboy Nanotechnology for detecting and eliminating cancer cells Objectives: Understand key concepts of the nanotechnology platforms that are designed for anti‐ cancer treatments Biological fundamentals: Cancer stem cells, CD47, Antibody, FAB fragment, Phage display, yeast display, ribosome display oligonucleotide Major challenge is in uniquely identifying and targeting cancer cells, while sparing normal cells ??? DNA damagegenomic instabilityloss of “guardians of the genome”aneuploidyselection for growth and immune evasion immune evasion cancer cells Cancer stem cell Cancer stem cell Stem cell Normal cells Up‐regulation of certain molecules: “do not eat me”, e.g. CD 47 EGF receptor (MAPK signaling); Hypoxic; Dividing Oligonucleotide delivery Major challenges for oligonucleotide d l h ll f l l d delivery in vivo. After administration into blood circulation (1), the f d bl d l ( ) h targeted ON particles are exposed to plasma components that can degrade the ON molecules. Further, they need to survive the renal excretion in order to reach the target tissue. To reach the target cells, they must overcome the vasculature barrier through an extravasation process (2), avoidance of the lymphatic drainage, and penetrating the interstitium either by convection or by diffusion. The targeted l h d d h h b b d ff h d particles are further bound to cellular receptors, and transported into the cytoplasm through a receptor‐ mediated endocytosis process (3). Inside the cells, the ONs need to escape the endosome (4) and be released to the cytoplasm to reach the target mRNAs The AAPS Journal, Vol. 11, No. 1, March 2009 Ligand‐directed liposomal and polymer ON nanoparticles; The AAPS Journal, Vol. 11, No. 1, March 2009 Lyposome‐based delivery of antibodies, nucleic acid, etc. L b d d li f tib di l i id t http://www.memsuniverse.com/?p=1635 http://www memsuniverse com/?p=1635 Key Features to Molecular Design 1. Anti‐TGFβ1 antibody 2. Biocompatible Carrier System 3. Interface between carrier and bioactive molecule Interface between carrier and bioactive molecule Antibody Fab fragment PLGA Nanoparticle ON Covalent Linkage Site‐specific Protein Modification • Chemoselective – Reacts specifically with protein N‐terminus • Orientation Control – Site specific modification Site‐specific modification allows control of orientation • Bioactive – Normal epitope binding of IgG and Fab Reacts with alkoxyamine Reacts with alkoxyamine (–ONH2) Scheck et. al., ACS 2007 Biotechnol. Appl. Biochem. (2009) 53, 1–29 ( The Y-shaped IgG molecule consists of two identical light chains ( ) and two identical heavy chains ( ) p g g (L) y (H) held together by disulfide bonds. Both the light and the heavy chain consist of a variable (V) (indicated in light grey) and a constant part (C) (indicated in white). On the tip of the arms, the variable regions of the heavy and light chains combine to form two identical antigen-binding sites containing six hypervariable loops, referred to as CDRs (indicated as thick black lines). The stem of the Y-shaped IgG, the Fc, is responsible for recruiting different effector functions and can provide longer half lives through interactions half-lives with Fc receptors. Also shown are different extensively investigated antigen-binding antibody fragments, namely Fab 2 fragment, Fab fragment, scFv fragment, diabody, minibody and dAb (of either variable heavy or light chain). Biotechnol. Appl. Biochem. (2009) 53, 1–29 ( Selection platforms (A) The three most established selection platforms: phage display, yeast display and ribosome display. (B) First, library diversity is generated from synthetic genes or shuffling procedures. The size typically ranges from 107 to 1013 variants. Next, the genes of the library are attached into/on to a molecular carrier (e.g. phage, yeast or ribosome) via a genotype-phenotype link. After translation of the gene (wavy line) to a protein (boxes in green, red and blue), each host particle will display a unique protein and encapsulate the corresponding encoding DNA. Next, the library encounter immobilized antigen. The particles displaying a binding protein that can recognize the antigen with sufficient affinity under the selection pressure will remain bound and the non-binding particles are removed by washing. Retained binders with desirable properties can be enriched by repeating the selection process after amplifying the eluted binders or genes. Individual binding molecules are then subjected to screening procedures. Biotechnol. Appl. Biochem. (2009) 53, 1–29 ( Multiple microenvironments form in tumors because of in tumors because of concentration gradients around sparse blood vessels. Nutrients and oxygen extravasate from the blood lumen (red region), through bl d l ( d i ) th h the endothelial vessel lining (purple), and diffuse into the interstitial tissue. Close to vessels, , cells are viable and proliferating. Far from vessels, hypoxia and nutrient depletion create regions of necrosis. Large inter‐vessel of necrosis Large inter vessel distances also reduce the concentration of blood‐borne chemotherapeutics in distal tissue. Curr Opin Biotechnol. 2008 October ; 19(5): 511–517. Three mechanisms used to control therapeutic delivery include temporally controlled cytotoxin release, enzyme drug controlled cytotoxin release enzyme drug activation, and biomolecule secretion. Top) Bacteria located within a radiation field (yellow curves) express and secrete (dashed arrows) a cytotoxic compound (purple shapes), while non‐irradiated bacteria do not, illustrating how irradiation can be used to spatially and temporally can be used to spatially and temporally control delivery. Middle) Bacteria expressing (dashed arrows) an enzyme gene (dark blue shapes) are used for drug activation. The pro‐ drugs (light blue diamonds) enter tumors d (li h bl di d) (tan) via the bloodstream (red region) and are subsequently converted to the active drug (purple triangles). Bottom) Many g (p p g ) ) y biologically active compounds (blue squares), including antibodies (blue “Y” figures) and DNA (black curves) can be engineered for bacterial expression and secretion for bacterial expression and secretion (dashed arrows). Curr Opin Biotechnol. 2008 October ; 19(5): 511–517. Gold nano‐particles conjugated to antibodies against EGF receptor http://www.memsuniverse.com/?p 1319 http://www.memsuniverse.com/?p=1319 The use of magnetic nanoparticles in anti‐cancer treatment http://www.memsuniverse.com/?p=361 Check your understanding: You should be able to design a nanoparticle‐based anti‐cancer therapy, that selectively targets cancer cells while largely sparing normal cells. You should be able to use molecular evolution approaches to enhance the efficiency of the bioactive parts of such anti‐cancer nanoparticles y p p ...
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