1. 1 THERAPEUTIC AGENTS 1.Introduction 2.Recombinant Proteins 3.Nucleic Acids

2. 2 Therapeutic Agents Before the advent of molecular biotechnology most human proteins were available in only small (limited) quantities. Today hundreds of genes (~1000) for human proteins have been cloned, sequenced, expressed in the host cells and are being tested as therapeutic agents (drugs) in humans. Over 140 biopharmaceuticals on the market; over 400 in clinical trials Biopharmaceuticals include: – Proteins (made in bacterial, fungal or mammalian cell culture) (blockbuster drugs) erythropoietin (EPO) insulin interferon (Intron A) granulocyte-colony stimulating factor (G-CSF) human growth hormone (HGH, human somatotropin) tissue plasminogen activator (tPA) – Monoclonal antibodies (made in mammalian cell culture) – Vaccines live and inactivated viruses and bacteria subunit vaccines recombinant vaccines – Gene Therapy Products (viral and non-viral) Introduction

3. 3 Process of Drug Production Cells and plasmid + Cell line TransfectionCell culturePurification Drug substance (crude) Drug substance (pure) Drug product - (sterile) Formulation/ Filling Cell line manufacture Medium development Bioreactor process development & scale-up Downstream purification Analytical characterization Introduction Therapeutic Agents

4. 4 Development of the Process Cell Line/ Viral Vector/ Construction Cell/Virus Culture Development/ Media Optimization Purification Process Dev. Formulation Design/ Drug Delivery Design Biological Assay Development and Support Facility/Equipment Design Technology Transfer Process Validation Pilot-Scale cGMP Production Commercial-Scale Production Therapeutic Agents Introduction

5. 5 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections -> Scientists discovered an antiviral protein in 1957 that inhibited growth of influenza virus in chicken embryos. It was named interferon because it interfered with the growth of influenza virus. Anti viral proteins released by host cells (part of the immune system) Interfere with viral multiplication Host cell specific but not virus specific Different types of cells in animals produce different interferons

6. 6 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections 3 types of human interferon: – alpha interferon (13 genes) – beta interferon (2 genes) – gamma interferon (1 gene) Alpha & beta usually produced early in viral infections (viruses or viral RNA) - Gamma appears later -> Presence of double-stranded RNA indicates cell is infected -> Viral infected cells release alpha and beta interferons Diffuse to neighboring cells -> Virus can’t replicate

7. 7 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Antiviral Treatment: Interferon therapy – Limited lifetime, short lasting effect – Recombinant interferons Pure and fast Hybrid genes for enhanced/new activity – Oral administration

8. 8 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Why are Side Effects Common and Severe for Injectable Interferon? Injectable interferon (beta) is approved world-wide (FDA) for the treatment of various cancers and viral diseases. Interferon is a protein readily eliminated from the blood by the kidney. To counteract the kidney’s clearance of interferon from the blood injectable interferon must be given in doses much higher than what occur naturally. Side effects include flu-like symptoms, poor results on liver function tests, and blood cell abnormalities. More serious side effects include depression, epileptic seizures, or liver problems. Why is Oral Interferon Different? Low-dose oral interferon is given in doses 10 thousand times less than injectable interferon. Therefore, side effects are dramatically reduced. Oral interferon is human interferon alpha administered in a small tablet (lozenge) to humans or in powder to animals. Oral interferon binds to surface (mucosal) cells in the mouth and throat resulting in stimulation of white blood cells and activates hundreds of genes affecting the immune system in the peripheral blood of man, cattle and mice. Studies show oral interferon is effective against disorders such as cancer, viral diseases and autoimmunity.

9. 9 Mechanism of Action Interferon placed in the mouth binds to receptors in the mucosal lining and initiates systemic effects on the immune system in animals and man. These immunomodulatory effects are safe and effective in helping control viral and autoimmune diseases and cancer. IFN  Oral Epithelial Cells Tonsil Mandibular Lymph Nodes Activation of Cell Mediated Immunity Virus Activation of Humoral Immunity (Antibody) Activation of Perioral Lymphoid Cells and Peripheral Lymphoid Tissues Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections

10. 10 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Manufacturing Steps for Interferon:

11. 11 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections Human diseases in which oral interferon has been tested and reported to be safe

12. 12 Therapeutic Agents Recombinant Proteins Human Interferons -> to fight viral infections 14,000 people participated in controlled studies of placebo versus interferon treatment during a natural outbreak of Hong Kong influenza. Interferon (about 128 units) or placebo was dripped into the nose daily for 5 days starting about the time of the first reported influenza cases. Interferon significantly (P<0.01) reduced the number of influenza cases. Human Study – Influenza and Interferon Efficacy of Human Leukocyte Interferon as Prophylaxis Against Influenza 0 5 10 15 20 25 AdultsChildren 7-12 yr Children 2-6 yr Population (14,000 subjects total) Percent of Patients Sick Interferon Placebo Soloviev, Bull. WHO 41:683-688, 1969.

13. 13 Therapeutic Agents Recombinant Proteins Strategies for Optimisation of Recombinant Production Screening libraries of recombinant genes (IFNs, human growth hormone, TNF-a…) Screening of recombinant expression systems (E coli, fungi, Mammalian cells…) Delivery by intestinal bacteria (lactobacilli)

14. Hepatitis C Hepatitis C virus infection is one of the most common causes of liver diseases, which affects nearly 200 million people worldwide. Therapeutic agents that maximize the early antiviral response and maintain viral suppression throughout the course of therapy have the best chance of achieving lasting eradication of the virus from an infected individual. 14

15. Hepatitis C virus infection Affects nearly 200 million people worldwide Effective treatment is the combined use of the antiviral chemical compound ribavirin and IFN-α. Longer acting IFNs are required. Creating longer acting IFNs include: 1.Pegylation of the therapeutic agent 2. Producing albumin-IFN hybrid molecule( approved for human use) 15

16. One effective treatment for hepatitis C includes the combined use of antiviral chemical compound ribavirin and IFN-α. Longer lasting IFNs are needed so that the side effects from IFNs can be minimized,e.g.by lowering the required dosage and decreasing the required frequency of the treatment. Two approaches: 1.pegylation of the therapeutic agent. 2.Fuse IFN gene with inert and stable human serum albumin which will produce albumin- IFN hybrid protein. 16

17. Native IFN levels in the blood of a treated patient typically decrease rapidly,so 2 days after administration,they are undetectable. But the albumin-IFN hybrid molecule,the drug remains in the serum at a therapeutically effective level for a much longer time. Recently approved for human use. 17

18. 18

19. Human Growth Hormone One of the first reombinant therapeuti proteins in the world to be approved for human use. First recombinant growth hormone was somatrem( produced and marketed by Genenteh). Sequene was identical to human growth hormone(exception:extra methionine residue at N terminal) Due to short half life in plasma HGH requires subutaneous injection once a day. 19

20. To prduce long lasting HGH DNA enoding the extraellular domain of the human growth hormone reeptor was fused to human growth hormone cDNA using a DNA fragment encoding a 20 amino acid long linker peptide consisting of four repeats of the amino aids Gly4Ser. 20

21. 21

22. Tumor Nerosis Factor 212 amino acid long transmembrane protein arranged in stable homotrimers that are subseuently cleaved by metalloprotease to form soluble homotrimeric cytokine TNF. Potent antumour agent. To develop TNF with tumor : DNA enoding short peptide (cys-asn-gly-arg-s-gly: targets tumor cell surface protein) was fused to TNF DNA. Modified TNF was 12 to 15 times more effetive at inhibiting tumor growth than the unmodified form. 22

23. Engineered Bacteriophage 23