Medical biotechnologyMedical biotechnology

Recombinant protein based Recombinant protein based pharmaceuticals and diagnosticspharmaceuticals 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 thenatural 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 EPOis dangerously abused by some athletes.

Recombinant proteins – eukaryotic systems

The first cloned mammal, Dolly wascreated 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 epitopesof an antigenic molecule

Clones of one single cells secreteidentical antibody moleculesrecognizing one single epitope

Monoclonal antibodiesProduction of monoclonal antibodies

Spleen cells of an immunized animalsecrete antibodies but have a short

life. Myeloma cells are ‘immortal’.

Hybrides of these cells (hybridoma cells)

can be immortal and we can selectthose 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' antibodiesAntibodies 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 domainwe get a humanized antibody

A 'humanized' antibody behave as a full human antibody.

Immunizing transgenic mice carrying human BCR (Ig) genes we cancreate fully human antibodies without genetic engineering.

'Humanized' antibodiesAntibodies 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 domainwe get a humanized antibody

A 'humanized' antibody behave as a full human antibody.

Immunizing transgenic mice carrying human BCR (Ig) genes we cancreate 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 substancesform ‘magic bullets’.

If effector functions (complement activation, opsonisation) aredispensable, 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

• 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-VEGFmonoclonal, 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

• 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

Histology

Immunoprecipitation

Cell separation

Protein (pathogen) identification

Affinity chromatography

Tumor diagnostics

Tumor therapy

RIA, ELISA

Immunogold ‘staining'

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

• 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 chromato-graphy

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.