Untitled Flashcards Set

PHAL 241: Proteins as Drugs lyn.wise@otago.ac.nz Small molecule drug Sialic acid 0.2 kDa Protein drug IgG1 antibody 150 kDa Objectives •Outline why proteins are useful as drugs. •Describe how recombinant antibodies and proteins are made for use as drugs. • Consider the advantages & disadvantages of proteins as drugs. Proteins • Catalyse biochemical reactions. • Form receptors and channels in membranes. • Provide scaffolding support. • Transport molecules within a cell and between organs. • Provide communication between cells and organs. Functions of Proteins • Disease can result when proteins: • Contain mutations or other abnormalities. • Are present in an abnormally high or low concentration. • Therapeutically, we can target or use proteins to alleviate disease. Proteins in Disease • In the 1880s, Emil von Behring, a German doctor, developed a serum (antibody) therapy from horses that neutralised diphtheria toxin. • For which he received the first Nobel Prize in Medicine in 1901. First Protein Therapy Antibodies • Large proteins produced by immune cells in response to detecting foreign antigens . • Directly bind to and block the action of that antigen. • Also triggers elimination of the antigen by immune cells. • Antibody therapies be used to eliminate disease -causing proteins. Heavy constant chain = Fc Light chain = LC Antigen binding Immune -cell activation Heavy determinant chain = Fd Making Antibodies • In the body, B cells recognise foreign antigens through surface antibodies • The antigen is presented to T cells which release cytokines to activate more B cells • B cells expand in number and change into memory B cells and plasma cells • Plasma cells secrete antibodies that target the original antigen Making Antibodies in the Lab • Hybridoma technology discovered in 1970s by Kohler and Milstein (Nobel Prize in 1984). • Antibody-producing B cells fused with cancer cell line in lab. • This enabled production of unlimited quantities of monoclonal antibodies against any protein target. • The first antibody drug was muromonab - OKT3. • Mice were immunised against human T cells, with antibodies against the cells (CD3) identified by screening hybridomas. • Produced at large scale in the lab, purified using chromatography. • Approved for clinical use in 1986, • To prevent rejection following organ transplantation. First Lab-made Antibody Therapy • Canadian doctor Frederick Banting, and colleagues, were first to isolate the hormone insulin in the 1920s. • Showed that a pancreatic extract reduced blood sugar levels in a diabetic dog. • Insulin was purified from animal pancreases then injected to patients in diabetic comas (Nobel Prize in 1923). • But tissue availability, cost, and immune reactions limited its clinical use. Second Protein Therapy Insulin Replacement Therapy • Protein produced by beta cells in the pancreas in elevated blood sugar. • Causes cells in the liver, muscle, and fat tissue to take up glucose and convert it to glycogen which can be stored in liver and muscles. • Insulin production is reduced in diabetes mellitus. • Insulin therapy could therefore be used to restore healthy levels of this protein. Insulin monomer Zinc Making Insulin in the Lab • Recombinant DNA technologies, developed from the 1970s, enabled any gene of interest to be cloned then expressed at high levels in other cells. • First recombinant human insulin protein produced was Humulin in 1979, which was approved for clinical use in 1982. • Still available in many injectable forms. • Most therapeutic antibodies and proteins are now produced recombinantly in bacterial, yeast or mammalian expression systems. Recombinant DNA Technologies • Recombinant DNA technologies enabled lab production of chimeric, humanised and human monoclonal antibodies. • LC, Fd and Fc domains are cloned from animal and/or human antibodies, then produced from mammalian cells. New & Improved Antibodies – expand this Recombinant Antibody Technologies • Bioreactors used for large scale production. • Special culture medias and growth conditions needed to grow large batches of protein/antibody producing cells. • These must then be purified from the cells or their media. Recombinant Protein Technologies Antibody Purification • Serum or cell media contain antibodies and mixture of other biological materials. • Antibodies are purified using chromatography with beads containing protein A (from Staphylococcus aureus). • Unwanted molecules are washed out, then purified antibodies are eluted off the beads. Protein Purification • Secreted proteins are collected in cell media, intracellular proteins need cell lysis first. • Both will contain a mixture of other biological materials. • Proteins can require more than one chromatography technique to purify – depends on protein structure. New & Improved Antibodies – expand this Bioprocessing • Protein drugs require many rounds of filtration to remove cellular and microbial contaminants. • Each step increases the time and costs of manufacturing. • Collectively known as biologics. • Most are monoclonal antibodies and their conjugates. • Others are hormones, enzymes, and other proteins. Biologics Pros and Cons of Biologics Successful Drug Target - TNF • The anti-cancer effects of tumor necrosis factor (TNF) were first described in 1962. • TNF protein was identified and cloned in 1985. • Produced by cells in response to tissue damage: • If in excess, can activate caspase cleavage to promote apoptosis. • But also activates NF-kB pathways to promote inflammation. • TNF inhibitors explored for treatment of inflammatory disease. TNF ↑ inflammation ↑ cell infiltration ↑ angiogenesis ↑ cell death Psoriasis Rheumatoid arthritis Inflammatory bowel disease Post-viral syndrome Anti-TNF biologics • Anti-TNF monoclonal antibodies: • Human/humanised is less immunogenic/safer than chimeric or mouse. • Truncated fusion to PEG offers improved safety. • Recombinant TNF receptors: • PEG improves half-life. • Antibody fusion improves targeting and delivery. human IgG1 mouse Fv humanised Fv human TNFR2 human Fc human TNFR1 PEG human LC/Fd PEG Infliximab Adalimumab Etanercept Onercept Certolizamab Endless Targets • Proteins serve highly specific and complex functions that cannot be replicated by simple chemical compounds. • Proteins and antibodies are produced from living organisms, and in the lab using recombinant technologies. • As drugs, proteins are complex to produce and purify, which makes them expensive to use. • But they can safely be used to block or destroy, replace or restore, or regulate proteins implicated in ANY disease. IMPORTANT: what to remember Up next: Friday 1 pm: Lecture Tutorial – Drug Discovery – opportunity for feedback on posters See you then!