Recombinant DNA in Medicine Industry- Monoclonal Antibodies Topics in Nanobiotechnology- April 2004-...

Recombinant DNA in Medicine Industry- Monoclonal Antibodies

Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte

Introduction


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

Expression Systems are developed to Produce Recombinant Proteins


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


The choice of which cell is used dependes on the project goal and on the properties of the protein to be produced

Expression system

Positive Negative

Bacterial cells(B. subtilis)

SimplicityShort generation timesLarge yields of productCan be induced to secrete the product into the culture mediumEj: 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 bodiesForeign proteins are sometimes toxic to bacteriaLack 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 canCan be induced to secrete the product into the culture mediumPerform posttranslational modifications

Active proteases that degrade foreign proteins (reducing yield of product)

Insects cell by baculovirus vectors

High level expressionCorrect foldingPosttranslational modificationsEj: 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

CostStill in development

Expression Systems

Monoclonal Antibodies Function


•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

Researchers tested myelomas


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

Hybridoma


Recombinant DNA- Watson, Gilman, Witkowski, Zoller. Scientific American Books. 1992



Bypassing fusion step

Direct cloning antibody cDNAs from the lymphocytes of immunized mice

More Monoclonal antibodyes


Humanized MAb

Bispecific antibodies

Effector Domains modification

More Monoclonal antibodyes- Humanized


Humanized monoclonal antibodies

Monoclonal antibodies Usually mouse protein

What about this foreing proteins in human system?



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




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



Review articleSupported by a grant from Zeneca PharmaceuticalsHumanized antibodies as potentialtherapeutic drugsSurender K Vaswani, MD and Robert G Hamilton, PhD



Bispecific antibodies


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. [email protected]

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

Thanks for your attention


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