Chapter 10-Protein Therapeutics

•Pharmaceutical proteins and enzymes•Monoclonal antibodies and recombinant antibodies

Table 10.1 Some recombinant proteins approved for human use ($60 billion-2006)

Protein Company Disorder

Factor VIII Baxter, Bayer Hemophilia A

Factor IX Genetics Institute Hemophilia B

Tissue plasminogen activator (TPA)

Genetech Acute myocardial infarction

Insulin Eli Lilly, Novo Nordisk Diabetes mellitus

Human growth hormone

Eli Lilly, Genetech, Upjohn, Novo Nordisk

GH deficiency in children (dwarfism)

Erythropoietin Amgen, Ortho Biotech Anemia

DNase I Genetech Cystic fibrosis

Various interferons (IFN)

Schering, Biogen, Chiron,Genetech

Hepatitis B and C, multiple sclerosis

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Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Table 10.1

Recombinant proteins-from http://en.wikipedia.org/wiki/List_of_recombinant_proteins -9/10/12

Human recombinants that largely replaced animal or harvested from human types• Human growth hormone (rhGH) Humatrope from Lilly and Serostim from Serono replaced cadaver

harvested human growth hormone• Human insulin (rhI) Humulin from Lilly and Novolin from Novo Nordisk among others; largely replaced

bovine and porcine insulin for human therapy. Some prefer to continue using the animal-sourced preparations, as there is some evidence that synthetic insulin varieties are more likely to induce hypoglycemia unawareness. Remaining manufacturers of highly-purified animal-sourced insulin include the U.K.'s Wockhardt Ltd. (headquartered in India), Poland's Polfa Tarchomin S.A., Argentina's Laboratorios Beta S.A., and China's Wanbang Biopharma Co.

• Follicle-stimulating hormone FSH replaced Serono's Pergonal which was previously isolated from post-menopausal female urine

• Factor VIII Kogenate from Bayer replaced blood harvested factor VIIIHuman recombinants with recombination as only source• Erythropoietin (EPO) Epogen from Amgen• Granulocyte colony-stimulating factor (G-CSF) filgrastim sold as Neupogen from Amgen; pegfilgrastim sold as

Neulasta• alpha-galactosidase A Fabrazyme by Genzyme• alpha-L-iduronidase (rhIDU; laronidase) Aldurazyme by BioMarin Pharmaceutical and Genzyme• N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Naglazyme (TM) by BioMarin Pharmaceutical• DNAse Pulmozyme by Genentech• Tissue plasminogen activator (TPA) Activase by Genentech• Glucocerebrosidase Ceredase by Genzyme• Interferon (IF) Interferon-beta-1a as Avonex from Biogen Idec; Rebif from Serono; Interferon beta-1b as

Betaseron from Schering• Insulin-like growth factor 1 (IGF-1)Animal recombinants• Bovine somatotropin (bST)• Porcine somatotropin (pST)• Bovine Chymosin

Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein. Host cells include :Bacteria - e.g. Escherichia coli (E.coli), Bacillus subtilis (B. subtilis)Yeast Cultured insect cells Cultured mammalian cells

The choice of cell type used depends upon the protein to be expressed. All require DNA to be cloned into the an appropriate vector.Advantages of bacterial cells

simple physiology short generation times, as bacteria grow and multiply rapidly large yields of product - up to 10 % of mass (low cost)

With B. subtilis and some others, it is possible to induce secretion of a gene product into the surrounding medium. This method is in use in the pharmaceutical industry in the production of hormones such as insulin and human growth hormone.Disadvantages of bacterial cells

The expressed proteins often do not fold properly and so are biologically inactive.The synthesised protein is often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is to incorporate an inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown Lack of enzymes responsible for post-translational modifications (effect on function of proteins), eg if the protein to be expressed is a glycoprotein, there is not apparatus in the bacterium to 'stick on' the necessary sugar residues.

Advantages of yeast cellsYeast is a simple eukaryote and performs many of the post-translational modifications required for human proteins Can be induced to secrete certain proteins into the growth medium for harvesting - e.g. Hepatitis B virus (HBV) vaccine.

Disadvantages of yeast cellsPresence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this problem is the construction of yeast strains from which the protease genes have been deleted).

Insect Cells-Expression of foreign proteins in insect cells through incorporation of their genes into baculovirus vectors Advantages of insect cells

High level of expression Correct folding Post-translational modifications similar to those in mammalian cells Cost, though more than for culturing bacteria and yeast, less than for mammalian cells e.g. potential vaccine for AIDS virus produced by expression of one of the HIV glycoproteins with this system

Disadvantages of insect cellsMore difficult to work with Expensive Slow generation time Not suitable for proteins with repetitive sequences

Cloning and expression of a foreign protein in a suitable host

Use of an appropriate expression vector and host

Example: A simple E. coli expression vector utilizing the lac promoter. (a) The expression vector plasmid contains a fragment of the E. coli chromosome containing the lac promoter and the neighboring lacZ gene. In the presence of the lactose analog IPTG, RNA polymerase normally transcribes the lacZ gene, producing lacZ mRNA, which is translated into the encoded protein, -galactosidase. (b) The lacZ gene can be cut out of the expression vector with restriction enzymes and replaced by the Granulocyte-Colony Stimulating Factor G-CSF cDNA. When the resulting plasmid is transformed into E. coli cells, addition of IPTG and subsequent transcription from the lac promoter produces G-CSF mRNA, which is translated into G-CSF protein.

Table 10.3 Some therapeutic monoclonal antibodies approved for human use

Type of antibody Company Therapeutic use

Mouse, Humanized

Ortho Biotech, Protein Design, Hoffmann-LaRoche

Prevents kidney transplant rejection

Chimeric Centocor Prevents blood clots

Chimeric Genetech, Hoffmann-LaRoche

Non-Hodgkin lymphoma

Humanized (Herceptin)

Genetech HER2-positive breast cancers

Humanized Am Home Prod, Celltech, Schering, Millen. Pharm.

Certain leukemias

Humanized Genetech Asthma

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Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Table 10.3

Antibody Structure•Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a "Y" shaped molecule. •The amino acid sequence in the tips of the "Y" varies greatly among different antibodies. This variable region, composed of 110-130 amino acids, give the antibody its specificity for binding antigen. The variable region includes the ends of the light and heavy chains. Treating the antibody with a protease can cleave this region, producing Fab or fragment antigen binding that include the variable ends of an antibody.

•The constant region determines the mechanism used to destroy antigen. Antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based on their constant region structure and immune function.

Copyright © 2010 ASM PressAmerican Society for Microbiology

1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Figure 10.24 Structure of an antibody molecule

Complementarity-determining regions (CDRs)

Copyright © 2010 ASM PressAmerican Society for Microbiology

1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Figure 10.25 Genetically engineered chimeric antibody

Copyright © 2010 ASM PressAmerican Society for Microbiology

1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Figure 10.26 Genetically engineered humanized antibody

Copyright © 2010 ASM PressAmerican Society for Microbiology

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Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Chapter 10Protein Therapeutics

Figure 10.40

Making monoclonal antibodies even more effective therapeutic agents: two ways