Transcript Orvosi biotechnológia Rekombináns fehérje alapú gyógyszerek

Medical biotechnology
Recombinant protein based
pharmaceuticals and diagnostics
Recombinant proteins –
prokaryotic systems
In vitro recombinant DNA technology (genetic engineering)
Transgenic or Genetically modified organisms (GMO)
Sequences of isolated genes recombined into new constructs, controlled by strong
promoters (enhancers). Expression vectors provide highly active genes.
Transforming cells with DNA construct we can create GMOs which synthesize
quantities of the gene product.
P
gene
Recombinant proteins –
prokaryotic systems
One of the first products of the bio-industry was the recombinant human insulin,
produced by transgenic E. coli cells
Recombinant insulin saves the life
of tens of millions of people
The molecule is identical with the
natural human insulin, no
immune reaction is triggered.
Recombinant proteins –
prokaryotic systems
During the last 15-20 years a number of recombinant proteins entered the
clinical practice. They are produced by transgenic bacteria.
insulin – diabetes
blood clotting factors VIII and IX – hemophilia
TPA (tissue plasminogen activator) – thrombosis, infarcts
human growth hormone – dwarfism,
interferons – virus infections, virus-induced malignancies
GM-CSF and IL-3 – leukopenia, bone marrow damage
angiostatin and endostatin – inhibition of angiogenesis in malignancies
ADA (adenosine deaminase) – inherited immunodeficiency
viral and bacterial antigens – vaccination, prevention of disease
Recombinant proteins –
eukaryotic systems
There are many proteins which can not be produced in the proper conformation by
prokaryotic cells. These are synthesized by eukaryotic (or mammalian) cells.
DNA constructs (shuttle vectors) are created in bacterial systems even in these
cases, but the transfromed cells are of eukaryotic origin.
Yeast cells
Insect cells / baculovirus
Animal (human) cell lines
Transgenic animals (milk)
erythropoetin,
blood clotting factorsa
HBV, virus proteins, etc.
Recombinant proteins –
eukaryotic systems
No red blood cells are produced in the absence of erythropoetin. EPO is produced
by the kidneys. Kidney patients, people on dialysis could survive only by
repeated transfusions.
EPO is produced by special yeast strains, which can modify the protein with proper
carbohydrate side chains.
Natural EPO was purified from the urine of certain anemic patients, but was not
available for clinical use. Now EPO
is dangerously abused by some
athletes.
Recombinant proteins –
eukaryotic systems
The first cloned mammal, Dolly was
created to produce more of the
flock secreting blood clotting
factors in their milk.
Biotechnology of antibodies
Polyclonal and monoclonal
antibodies
A number of different B cells
recognize different epitopes
of an antigenic molecule
Clones of one single cells secrete
identical antibody molecules
recognizing one single epitope
Monoclonal antibodies
Production of monoclonal antibodies
Spleen cells of an immunized animal
secrete antibodies but have a short
life. Myeloma cells are ‘immortal’.
Hybrides of these cells (hybridoma cells)
can be immortal and we can select
those ones which secrete antibodies.
These clones can be used to produce
large quantities of monoclonal
antibodies.
(Most of these hybrid cell lines
tend to be genetically unstable.)
'Humanized' antibodies
Antibodies are usually rised in rodents. These Ig molecules elicit an immune
response in humans and have very short half life.
Recognition of antigen is done by 3-3 fingers of the variable domains of light and
heavy chains. The rest of the molecule can be replaced with human sequences
without influencing antigen recognition. If the constant domains are human, we
have a chimaeric antibody.
Exchanging even the (non-recognizing)
sequences of the variable domain
we get a humanized antibody
A 'humanized' antibody behave as a
full human antibody.
Immunizing transgenic mice carrying
human BCR (Ig) genes we can
create fully human antibodies
without genetic engineering.
'Humanized' antibodies
Antibodies are usually rised in rodents. These Ig molecules elicit an immune
response in humans and have very short half life.
Recognition of antigen is done by 3-3 fingers of the variable domains of light and
heavy chains. The rest of the molecule can be replaced with human sequences
without influencing antigen recognition. If the constant domains are human, we
have a chimaeric antibody.
Exchanging even the (non-recognizing)
sequences of the variable domain
we get a humanized antibody
A 'humanized' antibody behave as a
full human antibody.
Immunizing transgenic mice carrying
human BCR (Ig) genes we can
create fully human antibodies
without genetic engineering.
Antibodies and derivatives
Monoclonal and bispecific antibodies
Fusion of two hybridoma cells of different specificities leads to the production of
bispecific antibodies. Antibodies
recognizing surface antigens of
tumor cells and toxic substances
form ‘magic bullets’.
If effector functions (complement
activation, opsonisation) are
dispensable, the Ig molecule can
be truncated (to double or single
chain Fab or Fv sequences).
Antibody derivatives
Tumor targeting ‘Magic bullets’ can be created from monospecific antibodies (or
derivatives) by chemically cross-linking toxic substances to Ig molecules.
These drugs are targeted, increasing efficiency and lowering side effects
Clinical use of antibodies
Pharmaceutical antibodies
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Pharmaceutical antibodies are produced
in high tech facilities under strict control
Development and registration of new
drugs takes many years and huge
investments
Pharmaceutical antibodies
•
Avastin blocks neovascularization,
proliferation of endothelial cells.
Avastin is a humanized anti-VEGF
monoclonal, with anti-tumor effect.
Remicade, Infliximab, Humira are neutralizing TNF. They are used agains reumatic
arthritis, Crohn disease, ulcerative colitis, autoimmune and chronic inflammatory
diseases.
In a similar way the anti-IL-1 monoclonal Anakinra is very effective in chronic
inflammatory conditions.
Lucentis (Ranimizumab) is used against macular degeneration, a primary cause of
blindness among elderly people.
Pharmaceutical antibodies
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Herceptin is a humanized monoclonal inhibiting dimerization of subunits of
EGF receptor.
EGF.R over-production (and a false signal) plays a significant role in many
cancers, especially in mammary carcinoma.
Xolair (Omalizumab) blocks IgE, easing
symptomes of severe asthma and allergy.
Pharmaceutical antibodies
• Colorectal cc. is treated by two other anti-EGF.R monoclonals: Vectibix
(Panitumumab) and Erbitux (Cetuximab).
Some viral diseases are also prevented by monoclonals, eg.:
respiratory syntitial virus (RSV) is neutrlized by Synagis (Palivizumab).
Host versus graft and graft versus host diseases can be prevented or treated
by Basiliximab or Daclizumab.
Non-Hodgkin limphoma can be successfully treated by Rituxan, (Tositumomab)
a monoclonal recognizing CD20, a proteins characteristic of B cells.
Diagnostic use of antibodies
Histology
Immunoprecipitation
Cell separation
Protein (pathogen) identification
Affinity chromatography
Tumor diagnostics
Tumor therapy
RIA, ELISA
Diagnostic use of antibodies
Immunogold ‘staining'
Histology
Immunoprecipitation
Cell separation
Protein (pathogen) identification
Affinity chromatography
Tumor diagnostics
Tumor therapy
RIA, ELISA
Fluor-labelled Igs
Diagnostic use of antibodies
Histology
Immunoprecipitation
Cell separation
Protein (pathogen) identification
Affinity chromatography
Tumor diagnostics
Tumor therapy
RIA, ELISA55
Diagnostic use of antibodies
Immunprecipitation
Detection of antibodies
Detection of antigens
Hemagglutination
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Quantitation of antigens / antibodies by immunoprecipitation / hemagglutination.
U or V shaped wells on microplates: RBC (or latex) pellet into small dots if not
precipitated. Immune reaction causes large spots of Ab-Ag networks
Diagnostic use of antibodies
Histology
Immunoprecipitation
Cell separation
Protein (pathogen)
identification
Affinity chromatography
Tumor diagnostics
Tumor therapy
RIA, ELISA
Magnetic beads
Diagnostic use of antibodies
Histology
Immunoprecipitation
Cell separation
Protein (pathogen) identification
Affinity chromatography
Tumor diagnostics
Tumor therapy
RIA, ELISA
FACS: fluorescence-activated cell sorter
Transgenic animals
To learn about the function of genes
transgenic animals with knocked out
genes or transgenic mice are extremely
important.
Most of our genes are practically identical
with mice genes, these experiments tell a
lot about our genes.