Transcript Recombinant DNA in Medicine Industry

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
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
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
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Expression Systems
Expression
system
Bacterial cells
(B. subtilis)
Positive
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)
Negative
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
The choice of which cell is used dependes on the project goal and on the
properties of the protein to be produced
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
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
Major limitation in the
therapeutic use of antibodies
Producing a useful antibody
in large quantities
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Researchers tested myelomas
Reserchers tested myelomas
Development of monoclonal
antibody technology
Lack to produce an antibody to
their specifications
Hybridoma- produce antibodies
specified by the lymphocyte
from the immunized animal
•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
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Hybridoma
Recombinant DNA- Watson, Gilman, Witkowski, Zoller.
Scientific American Books. 1992
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Bypassing fusion step
Direct cloning antibody cDNAs from
the lymphocytes of immunized mice
Recombinant DNA- Watson, Gilman, Witkowski, Zoller.
Scientific American Books. 1992
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
More Monoclonal antibodyes
Humanized MAb
Bispecific antibodies
Effector Domains modification
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
More Monoclonal antibodyesHumanized
Monoclonal antibodies
Usually mouse
protein
What about this foreing proteins in
human system?
Humanized monoclonal antibodies
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Humanized monoclonal
antibodies
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
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Humanized monoclonal
antibodies
Few of the hundred aminoacids in Variable region
contact the antigen (complementary determining
regions CDRs)
Just CDR to be transferred
Humanized MAb in clinical trials as an
immunosuppressant and for treatment of lymphoid
tumors.
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Humanized monoclonal
antibodies
Review article
Supported by a grant from Zeneca Pharmaceuticals
Humanized antibodies as potential
therapeutic drugs
Surender K Vaswani, MD and Robert G Hamilton, PhD
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Humanized monoclonal
antibodies
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
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]
http://users.telenet.be/nmertens/U11/IM_bispecific_antibodies.htm
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.
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte
Thanks for your attention
Topics in Nanobiotechnology- April 2004- Maria Viviana Duarte