Pharmaceutical Products of DNA Technology

Recombinant Proteins, Monoclonal Antibodies and Vaccines

Sherry Fuller-Espie, Ph.D., DIC Associate Professor Cabrini College Sherry Fuller-Espie, 2003

I. Introduction

A. The pharmaceutical industry produces a wide range of products using recombinant DNA techniques

Human proteins can be cloned

Gene recovered from human genome (as genomic DNA or cDNA) Placed in an expression vector using ligation techniques and restriction endonucleases Transform bacteria (e.g. Escherichia coli or Bacillus spp.) Grow in batch culture in large industrial scale vats Purify protein product

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

B. Therapeutics

Hormones or hormone-like compounds are used for human therapy Enzymes Antisense RNA

C. Vaccines generated through rDNA

Vaccinia virus as carrier of virus proteins Recombinant virus proteins made in yeast or bacteria Subtracting virulence genes from bacteria (e.g. cholera) DNA vaccines

II. Human Protein Replacements Can Treat Genetically-Linked Diseases

A. Insulin

Made by pancreatic beta cells Enables cells to take up glucose from the bloodstream to use in production of ATP Insufficient insulin causes diabetes (insulin dependent diabetes mellitus IDDM)

Must inject insulin to avoid physiological complications Cells cannot take up glucose Insufficient ATP is made Glucose 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)

Retinopathy Kidney failure Nerve disorders Circulatory 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 total

A chain = 21 amino acids B 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 processing

Preproinsulin

Contains a signal sequence + 3 sections of amino acids

Preproinsulin too difficult for E. coli to produce

Signal sequence is removed after targeting to RER Translation continues on RER > forming proinsulin Removal of 33 amino acids at Golgi Apparatus, and joining of A and B chains to form insulin

Simplification 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 differences Pig (Porcine) = 1 amino acid difference Amino acid differences can stimulate allergic responses Therefore 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 energy Dwarfism caused by insufficient production of HGH by the pituitary gland Growth retardation

HGH can treat dwarfism to help undersized children reach their normal height and size

Chubby face Baby fat around waist Unusual body properties as an adult ~ 4 feet tall only IQ = Normal

Old method:

Purification of HGH from cadaver pituitary glands

8 cadavers/year for 8 10 years per patient Risky to use brain tissue

Prion disease transmission: Creutzfeldt-Jacob Disease (CJD) Muscle wasting Convulsions Tremors Dementia 24 cases reported by 1993 in France from cadaver HGH

New method

Gene production for HGH synthesis

Protropin Genentech Humatrope Eli Lilly

C. Factor VIII

Hemophilia A affects 1/10,000 males in USA

Abnormal blood clotting in absence of Factor VIII Before rDNA, Factor VIII was obtained from blood
8000 pints needed per patient per year Risk of transmitting HIV before wide-spread screening for HIV

In the 1980s thousands of hemophiliacs were infected with HIV developed AIDS

Factor VIII genetic characteristics

Located on X chromosome 186 Kbp 26 exons, numerous introns Codes for 2332 amino acids

cDNA obtained from gene sequence and cloned into Hamster Kidney Cells Factor VIII protein

E. 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 FDA

Recombinate Genetics Institute Kogenate Miles Laboratories

Hypersensitivies observed in some patients

Perhaps due to glycosylation patterns Immunological 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
Fibrin

Plasmin
---------- Degradation of fibrin

Streptokinase once used for this purpose

Derived from Streptococcal bacteria Must be delivered to blood vessel directly Urokinase = alternative, but has risk of hemorrhage

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

Early 1980s Plasmid constructed

Shuttle vector had characteristics for maintenance in E. coli and expression in mammalian cells
cDNA for TPA TPA signal sequence TPA promoter TPA termination sites Antibiotic resistance + ORI for prokaryotic cells Methotrexate resistance marker for mammalian cells

Mammalian cells transfected Stable integration into genome of mammalian host cell High levels of secreted TPA produced in large scale culture conditions

FDA approves Activase Genetech 1987

Clot-Dissolving Agent Fewer side effects than streptokinase and urokinase

B. Interferon

a and b are induced by dsRNA in cells infected by viruses Bind to neighboring cells and send a warning signal of viruses nearby (speciesspecific receptors) Neighboring cells protect themselves by producing proteins that inhibit viral replication IFNs also activate Natural Killer (NK) cells

Before rDNA 90,000 pints of blood needed to purify 1 g IFNs 1980 recombinant a-IFN

Mammalian cell culture (glycosylated) Shown to:

Decrease symptoms of hepatitis Decrease spread of herpes zoster (shingles) Shrink certain tumors

Intron marketed in 1984 by Swiss Biotech Firm FDA approved its used in 1986 for use against a particular form of leukemia, and in 1988 for genital warts Also used to treat

Kaposis Sarcoma in AIDS patients Malignant melanoma Multiple myeloma Some kidney cancers

b-IFN 1b licensed in 1993 for Multiple Sclerosis

IV. Other Innovative Pharmaceuticals

A. Amgen Products

Erythropoietin

Kidney-derived hormone that stimulates RBC production in the bone marrow Epogen 1989 Recombinant hormone used to alleviate severe anemia that is a complication of many kidney diseases
Stimulates stem cells to produce neutrophils (and other leukocytes)

Neupogen

B. Cytokines

Cytokines = hormone-like substances that stimulate lymphocyte (and some leukocyte) activities

Interferons Colony-stimulating factors Interleukin-2 (T cell growth factor)

C. Monoclonal Antibodies

Specific antibodies produced in vitro which can bind to:

Cytokines (anticytokine Abs) Specific subsets of cells

Tumor cells

MoAb against cell-surface tumor Ags can be used for diagnosis and immunotherapy MoAb can be conjugated to toxins or radioactive isotypes to kill tumor cells = Immunotoxins

Attacking T cells in grafts

MoAbs can be humanized to eliminate hypersensitivity reactions (serum sickness)

Replace Fc portion of MoAb with human Fc portion Retain Fab which contains the antigenbinding site rDNA 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 humans Numerous animal models are under investigation for the use of DNA vaccines in humans

Malaria AIDS Herpes Tuberculosis Rotavirus (childhood diarrhea)

Differences between traditional vaccines and DNA vaccines

Just the DNA coding for a specific component of a disease-causing organism in injected into the body
Saline solution/hypodermic needle DNA-coated gold beads propelled into the body using gene guns

Production of the immunizing protein occurs in the vaccinated host Once inside cells, some of the rDNA go to the nucleus and instruct transcription of encoded antigenic proteins

Protein 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 surface Which 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 eliminated DNA 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 tolerance Random integration into the genome? Are plasmid vectors toxic? Does DNA delivered as a drug incite an immune response against the bodys own DNA?