1. Recombinant DNA in Medicine Industry- Monoclonal Antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte

2. Introduction Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte As soon as first successful cloning experiments were reported in 1973, applications for this powerful technology quickly followed- Proteins were produced through recombinant DNA technology for : Somatostatin (1976)- 14 aminoacids peptide neurtransmitter *Treatment of numerous diseases Insulin for the treatment of diabetes Human Growth Hormone Food Production MAb development etc

3. Expression Systems are developed to Produce Recombinant Proteins Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Cloning the gene or cDNA encoding a particular proteins is only the first of many steps needed to produce a recombinant protein Next step: put he gene into a host cell for production

4. Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte The choice of which cell is used dependes on the project goal and on the properties of the protein to be produced Expression system PositiveNegative Bacterial cells (B. subtilis)  Simplicity  Short generation times  Large yields of product  Can be induced to secrete the product into the culture medium Ej: Bacterial production of human growth hormone (hGH)  Although some proteins are expressed to high levels, they often the fail to fold properly and form insoluble inclusion bodies  Foreign proteins are sometimes toxic to bacteria  Lack enzymes that are present in eukaryotic cells and add posttranslational modifications (phosphates, sugars) Yeast  Simple eukaryote that resembles mammalian cells in may ways but can be grown as quickly and cheaply as bacteria can  Can be induced to secrete the product into the culture medium  Perform posttranslational modifications  Active proteases that degrade foreign proteins (reducing yield of product) Insects cell by baculovirus vectors  High level expression  Correct folding  Posttranslational modifications Ej: Vaccine for the AIDS virus has been prepared by producing on of the HIV glycoproteins with this system  Higher costs than bacteria and yeast, but lower than mammal cells Mammalian cells  Checking the function of a newly cloned gene and as a quick methods for assessing the function of engineered proteins.  Ej: large scale production for proteins such as tissue plasmoninogen activator  Cost  Still in development Expression Systems

5. Monoclonal Antibodies Function Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Antibodies are exquisitely selective proteins that can bind to a single target among millions of irrelevant sites Could effectively seek and destroy tumor cells and infectious agents wherever the reside Producing a useful antibody in large quantities Major limitation in the therapeutic use of antibodies

6. Researchers tested myelomas Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Lack to produce an antibody to their specifications Reserchers tested myelomas Hybridoma- produce antibodies specified by the lymphocyte from the immunized animal Development of monoclonal antibody technology Monoclonal antibodies are already widely used for the diagnostic of infections and cancer and for the imaging of tumors for radiotherapy Preparing specfic antibodies: abzyme- antibodies with catalytic activity, birecognition, etc

7. Hybridoma Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Recombinant DNA- Watson, Gilman, Witkowski, Zoller. Scientific American Books. 1992

8. Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Recombinant DNA- Watson, Gilman, Witkowski, Zoller. Scientific American Books. 1992 Bypassing fusion step Direct cloning antibody cDNAs from the lymphocytes of immunized mice

9. More Monoclonal antibodyes Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Humanized MAb Bispecific antibodies Effector Domains modification

10. More Monoclonal antibodyes- Humanized Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Humanized monoclonal antibodies Monoclonal antibodiesUsually mouse protein What about this foreing proteins in human system?

11. Humanized monoclonal antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte The variable regions differ in sequence from on antiblody to another, this is the region of the protein that binds the antigen First method: encoded proteins in which the variable regions from the mouse antibody were fused the constant regions from a human antibody Not fully humanized Recombinant DNA- Watson, Gilman, Witkowski, Zoller. Scientific American Books. 1992

12. Humanized monoclonal antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Humanized MAb in clinical trials as an immunosuppressant and for treatment of lymphoid tumors. Few of the hundred aminoacids in Variable region contact the antigen (complementary determining regions CDRs) Just CDR to be transferred

13. Humanized monoclonal antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Review article Supported by a grant from Zeneca Pharmaceuticals Humanized antibodies as potential therapeutic drugs Surender K Vaswani, MD and Robert G Hamilton, PhD

14. Humanized monoclonal antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte

15. Bispecific antibodies Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte Bispecific antibodies in cancer therapy. Segal DM, Weiner GJ, Weiner LM. Immune Targeting Section, Experimental Immunology Branch, National Cancer Institute, Building 10 Room 4B36, National Institutes of Health, Bethesda, MD 20892-1360, USA. dave_segal@nih.gov Based upon in vitro and animal studies, a number of Phase I and II clinical trials have been initiated to test whether bispecific antibodies could redirect immune effectors against tumor cells in cancer patients. Recently, results from those trials showed beneficial effects in some patients but it is clear many problems remain to be solved. In addition, molecular engineering approaches are providing new and improved sources of clinically relevant bispecific antibodies. http://users.telenet.be/nmertens/U11/IM_bispecific_antibodies.htm

16. Thanks for your attention Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte