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. Pgene

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 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 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 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.