Pharmaceutical Products of DNA TechnologyRecombinant Proteins, Monoclonal Antibodies and Vaccines

Sherry Fuller-Espie, Ph.D., DICAssociate ProfessorCabrini College Sherry Fuller-Espie, 2003

I. Introduction

A. The pharmaceutical industry produces a wide range of products using recombinant DNA techniquesHuman proteins can be clonedGene recovered from human genome (as genomic DNA or cDNA)Placed in an expression vector using ligation techniques and restriction endonucleasesTransform bacteria (e.g. Escherichia coli or Bacillus spp.)Grow in batch culture in large industrial scale vatsPurify protein product

Human proteins produce fewer side effects than proteins from other animals (e.g. pork insulin vs. human insulin)

B. TherapeuticsHormones or hormone-like compounds are used for human therapyEnzymes Antisense RNA

C. Vaccines generated through rDNAVaccinia virus as carrier of virus proteinsRecombinant virus proteins made in yeast or bacteriaSubtracting virulence genes from bacteria (e.g. cholera)DNA vaccines

II. Human Protein Replacements Can Treat Genetically-Linked Diseases

A. InsulinMade by pancreatic beta cellsEnables cells to take up glucose from the bloodstream to use in production of ATPInsufficient insulin causes diabetes (insulin dependent diabetes mellitus IDDM)Must inject insulin to avoid physiological complicationsCells cannot take up glucoseInsufficient ATP is madeGlucose spills into urine (excreted by kidneys; kidney tries to dilute glucose by excreting large amounts of water)

Complications of diabetes (60 x 106 worldwide cases)RetinopathyKidney failureNerve disordersCirculatory diseases (including gangrene and stroke)Diabetic coma (pH imbalance caused by fat metabolism producing ketones)

Insulin is made up of 2 chains = 51 amino acids totalA chain = 21 amino acidsB chain = 30 amino acids

S - S__l____________l____ A S S___S___________S____________________ B

Two disulfide bonds hold A and B together (interchain disulfide bond)One disulfide bond within the A chian (intrachai disulfide bond)Insulin processingPreproinsulinContains a signal sequence + 3 sections of amino acidsSignal sequence is removed after targeting to RERTranslation continues on RER > forming proinsulinRemoval of 33 amino acids at Golgi Apparatus, and joining of A and B chains to form insulinPreproinsulin too difficult for E. coli to produceSimplification had to be made!

Before recombinant insulin was available, insulin was obtained from cows or pigs pancreases (7-10 lb pancreatic tissue per patient per year)Cow (Bovine) = 3 amino acid differencesPig (Porcine) = 1 amino acid difference Amino acid differences can stimulate allergic responsesTherefore human insulin is preferred

B. Human Growth Hormone (HGH)HGH promotes overall body growth by increasing: amino acid uptake by cells, protein synthesis and fat utilization for energyDwarfism caused by insufficient production of HGH by the pituitary glandGrowth retardationChubby faceBaby fat around waistUnusual body properties as an adult~ 4 feet tall onlyIQ = NormalHGH can treat dwarfism to help undersized children reach their normal height and size

Old method:Purification of HGH from cadaver pituitary glands8 cadavers/year for 8 10 years per patientRisky to use brain tissuePrion disease transmission: Creutzfeldt-Jacob Disease (CJD)Muscle wastingConvulsionsTremorsDementia24 cases reported by 1993 in France from cadaver HGH

New methodGene production for HGH synthesisProtropin GenentechHumatrope Eli Lilly

C. Factor VIIIHemophilia A affects 1/10,000 males in USAAbnormal blood clotting in absence of Factor VIIIBefore rDNA, Factor VIII was obtained from blood 8000 pints needed per patient per yearRisk of transmitting HIV before wide-spread screening for HIVIn the 1980s thousands of hemophiliacs were infected with HIV developed AIDS

Factor VIII genetic characteristicsLocated on X chromosome 186 Kbp26 exons, numerous intronsCodes for 2332 amino acidscDNA obtained from gene sequence and cloned into Hamster Kidney Cells Factor VIII proteinE. coli NOT USED because protein needs extensive glycosylation (25 sites where CHO-groups are added to protein in ER and Golgi)

Factor VIII products approved by FDARecombinate Genetics InstituteKogenate Miles LaboratoriesHypersensitivies observed in some patientsPerhaps due to glycosylation patternsImmunological intolerance

III. Human Therapies

A. Tissue Plasminogen Activator (TPA)TPA is used to treat coronary thrombosis (thrombolytic agent)Protease that attaches to blood clots and induces other blood components to break down clot

**Plasminogen Plasmin

Fibrin ---------- Degradation of fibrinStreptokinase once used for this purposeDerived from Streptococcal bacteriaMust be delivered to blood vessel directlyUrokinase = alternative, but has risk of hemorrhage

TPA does not compromise blood clotting elsewhere therefore reduces risk of internal hemorrhagingAdministered IV transported via circulation to affected areaProduced in mammalian cell culture (not E.coli)

Early 1980s Plasmid constructedShuttle vector had characteristics for maintenance in E. coli and expression in mammalian cellscDNA for TPATPA signal sequenceTPA promoterTPA termination sitesAntibiotic resistance + ORI for prokaryotic cellsMethotrexate resistance marker for mammalian cellsMammalian cells transfected Stable integration into genome of mammalian host cellHigh levels of secreted TPA produced in large scale culture conditions

FDA approves Activase Genetech 1987Clot-Dissolving AgentFewer side effects than streptokinase and urokinase

B. Interferona and b are induced by dsRNA in cells infected by virusesBind to neighboring cells and send a warning signal of viruses nearby (species-specific receptors)Neighboring cells protect themselves by producing proteins that inhibit viral replicationIFNs also activate Natural Killer (NK) cells

Before rDNA 90,000 pints of blood needed to purify 1 g IFNs1980 recombinant a-IFN Mammalian cell culture (glycosylated)Shown to:Decrease symptoms of hepatitisDecrease spread of herpes zoster (shingles)Shrink certain tumors

Intron marketed in 1984 by Swiss Biotech FirmFDA approved its used in 1986 for use against a particular form of leukemia, and in 1988 for genital wartsAlso used to treat Kaposis Sarcoma in AIDS patientsMalignant melanomaMultiple myelomaSome kidney cancersb-IFN 1b licensed in 1993 for Multiple Sclerosis

IV. Other Innovative Pharmaceuticals

A. Amgen ProductsErythropoietinKidney-derived hormone that stimulates RBC production in the bone marrowEpogen 1989Recombinant hormone used to alleviate severe anemia that is a complication of many kidney diseasesNeupogenStimulates stem cells to produce neutrophils (and other leukocytes)

B. CytokinesCytokines = hormone-like substances that stimulate lymphocyte (and some leukocyte) activitiesInterferonsColony-stimulating factorsInterleukin-2 (T cell growth factor)

Monoclonal Antibodies

Specific antibodies produced in vitro which can bind to:Cytokines (anticytokine Abs)Specific subsets of cells Tumor cellsMoAb against cell-surface tumor Ags can be used for diagnosis and immunotherapyMoAb can be conjugated to toxins or radioactive isotypes to kill tumor cells = ImmunotoxinsAttacking T cells in grafts

MoAbs can be humanized to eliminate hypersensitivity reactions (serum sickness)Replace Fc portion of MoAb with human Fc portionRetain Fab which contains the antigen-binding siterDNA techniques using cDNA can be employed to cut and splice appropriate regions, followed by transfection into myreloma

Constant and Variable Domains of Antibodies: Location of constant (C) and Variable (V) domains within (a) light chains and (b) heavy chains. The dark blue bands represent hypervariable regions or complementarity-determining regions within the variable domains.

V. DNA Vaccines

DNA vaccine technology is showing increasing promise in the treatment of disease in humansNumerous animal models are under investigation for the use of DNA vaccines in humansMalariaAIDSHerpesTuberculosisRotavirus (childhood diarrhea)

Differences between traditional vaccines and DNA vaccinesJust the DNA coding for a specific component of a disease-causing organism in injected into the body Saline solution/hypodermic needleDNA-coated gold beads propelled into the body using gene guns

Production of the immunizing protein occurs in the vaccinated hostOnce inside cells, some of the rDNA go to the nucleus and instruct transcription of encoded antigenic proteinsProtein products can elicit humoral (Ab-type) IR if the proteins are released from the cells, or cell-mediated (killer-type) immunity is protein are processed/degraded intracellulary and properly displayed on the cell surfaceWhich cells take up rDNA is critically important for efficient recognition of antigen molecules (professional antigen presenting cells required for efficient T cell responses)

Risk of infection associated with live or attenuated virus vaccines is eliminatedDNA vaccination provides long-lived immune response (boosters not need to maintain immunity)Vaccines can be produced using similar techniques (simplification of development and production processes)Stable (dried or in solution)

As microbial genomes of pathogens are sequenced, the sequence information can be used for vaccine design Potential Risks:Potential for induction for toleranceRandom integration into the genome?Are plasmid vectors toxic? Does DNA delivered as a drug incite an immune response against the bodys own DNA?