Pharmaceutical Products of Pharmaceutical Products of DNA TechnologyDNA Technology

Recombinant Proteins, Monoclonal Antibodies and Vaccines

Sherry Fuller-Espie, Ph.D., DICAssociate Professor

Cabrini College© Sherry Fuller-Espie, 2003

I. IntroductionI. Introduction

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

DNA techniquesDNA techniques Human proteins can Human proteins can

be clonedbe cloned Gene recovered from Gene recovered from

human genome (as human genome (as genomic DNA or genomic DNA or cDNAcDNA))

Placed in an expression Placed in an expression vector using ligation vector using ligation techniques and techniques and restriction restriction endonucleasesendonucleases

Transform bacteria (e.g. Transform bacteria (e.g. Escherichia coliEscherichia coli or or Bacillus sppBacillus spp.).)

Grow in batch culture in Grow in batch culture in large industrial scale large industrial scale vatsvats

Purify protein productPurify protein product

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

B. TherapeuticsB. Therapeutics

Hormones or hormone-like Hormones or hormone-like compounds are used for human compounds are used for human therapytherapy

Enzymes Enzymes Antisense RNAAntisense RNA

C. Vaccines generated through C. Vaccines generated through rDNArDNA

Vaccinia virus as carrier of virus Vaccinia virus as carrier of virus proteinsproteins

Recombinant virus proteins made in Recombinant virus proteins made in yeast or bacteriayeast or bacteria

Subtracting virulence genes from Subtracting virulence genes from bacteria (e.g. cholera)bacteria (e.g. cholera)

DNA vaccinesDNA vaccines

II. Human Protein II. Human Protein Replacements Can Treat Replacements Can Treat

Genetically-Linked Genetically-Linked DiseasesDiseases

A. InsulinA. Insulin

Made by pancreatic beta cellsMade by pancreatic beta cells Enables cells to take up glucose from the Enables cells to take up glucose from the

bloodstream to use in production of ATPbloodstream to use in production of ATP Insufficient insulin causes diabetes (insulin Insufficient insulin causes diabetes (insulin

dependent diabetes mellitus – IDDM)dependent diabetes mellitus – IDDM) Must inject insulin to avoid physiological Must inject insulin to avoid physiological

complicationscomplications Cells cannot take up glucoseCells cannot take up glucose Insufficient ATP is madeInsufficient ATP is made Glucose spills into urine (excreted by kidneys; Glucose spills into urine (excreted by kidneys;

kidney tries to dilute glucose by excreting large kidney tries to dilute glucose by excreting large amounts of water)amounts of water)

Complications of diabetes (60 x 10Complications of diabetes (60 x 1066 worldwide cases)worldwide cases) RetinopathyRetinopathy Kidney failureKidney failure Nerve disordersNerve disorders Circulatory diseases (including gangrene Circulatory diseases (including gangrene

and stroke)and stroke) Diabetic coma (pH imbalance caused by Diabetic coma (pH imbalance caused by

fat metabolism producing ketones)fat metabolism producing ketones)

Insulin is made up of 2 chains = 51 Insulin is made up of 2 chains = 51 amino acids totalamino acids total A chain = 21 amino acidsA chain = 21 amino acids B chain = 30 amino acids B chain = 30 amino acids S - SS - S__l____________l____ A__l____________l____ A S SS S___S___________S____________________ B___S___________S____________________ B

Two disulfide bonds hold A and B together Two disulfide bonds hold A and B together (interchain disulfide bond)(interchain disulfide bond)

One disulfide bond within the A chian One disulfide bond within the A chian (intrachai disulfide bond)(intrachai disulfide bond)

Insulin processingInsulin processing PreproinsulinPreproinsulin

Contains a signal sequence + 3 sections of amino acidsContains a signal sequence + 3 sections of amino acids Signal sequence is removed after targeting to RERSignal sequence is removed after targeting to RER Translation continues on RER –> forming proinsulinTranslation continues on RER –> forming proinsulin

Removal of 33 amino acids at Golgi Apparatus, and Removal of 33 amino acids at Golgi Apparatus, and joining of A and B chains to form insulinjoining of A and B chains to form insulin

Preproinsulin too difficult for Preproinsulin too difficult for E. coliE. coli to to produceproduce Simplification had to be made!Simplification had to be made!

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

allergic responsesallergic responses Therefore human insulin is preferredTherefore human insulin is preferred

B. Human Growth Hormone (HGH)B. Human Growth Hormone (HGH)

HGH promotes overall body growth by increasing: HGH promotes overall body growth by increasing: amino acid uptake by cells, protein synthesis and amino acid uptake by cells, protein synthesis and fat utilization for energyfat utilization for energy

Dwarfism caused by insufficient production of Dwarfism caused by insufficient production of HGH by the pituitary glandHGH by the pituitary gland

Growth retardationGrowth retardation Chubby faceChubby face ““Baby fat” around waistBaby fat” around waist Unusual body properties as an adultUnusual body properties as an adult ~ 4 feet tall only~ 4 feet tall only IQ = NormalIQ = Normal

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

Old method:Old method: Purification of HGH from cadaver pituitary Purification of HGH from cadaver pituitary

glandsglands 8 cadavers/year for 8 – 10 years per patient8 cadavers/year for 8 – 10 years per patient Risky to use brain tissueRisky to use brain tissue

Prion disease transmission: Creutzfeldt-Jacob Prion disease transmission: Creutzfeldt-Jacob Disease (CJD)Disease (CJD)

Muscle wastingMuscle wasting ConvulsionsConvulsions TremorsTremors DementiaDementia

24 cases reported by 1993 in France from cadaver 24 cases reported by 1993 in France from cadaver HGHHGH

New methodNew method Gene production for HGH synthesisGene production for HGH synthesis

Protropin Protropin Genentech Genentech Humatrope Humatrope Eli Lilly Eli Lilly

C. Factor VIIIC. Factor VIII

Hemophilia A affects 1/10,000 males Hemophilia A affects 1/10,000 males in USAin USA Abnormal blood clotting in absence of Abnormal blood clotting in absence of

Factor VIIIFactor VIII Before rDNA, Factor VIII was obtained Before rDNA, Factor VIII was obtained

from blood from blood 8000 pints needed per patient per year8000 pints needed per patient per year Risk of transmitting HIV before wide-spread Risk of transmitting HIV before wide-spread

screening for HIVscreening for HIV In the 1980s – thousands of hemophiliacs were In the 1980s – thousands of hemophiliacs were

infected with HIV infected with HIV developed AIDS developed AIDS

Factor VIII genetic characteristicsFactor VIII genetic characteristics Located on X chromosome Located on X chromosome 186 Kbp186 Kbp 26 exons, numerous introns26 exons, numerous introns Codes for 2332 amino acidsCodes for 2332 amino acids

cDNA obtained from gene sequence and cDNA obtained from gene sequence and cloned into Hamster Kidney Cells cloned into Hamster Kidney Cells Factor Factor VIII proteinVIII protein E. coli NOT USED because protein needs E. coli NOT USED because protein needs

extensive glycosylation (25 sites where CHO-extensive glycosylation (25 sites where CHO-groups are added to protein in ER and Golgi)groups are added to protein in ER and Golgi)

Factor VIII products approved by FDAFactor VIII products approved by FDA Recombinate – Genetics InstituteRecombinate – Genetics Institute Kogenate – Miles LaboratoriesKogenate – Miles Laboratories

Hypersensitivies observed in some Hypersensitivies observed in some patientspatients Perhaps due to glycosylation patternsPerhaps due to glycosylation patterns Immunological intoleranceImmunological intolerance

III. Human TherapiesIII. Human Therapies

A. Tissue Plasminogen Activator A. Tissue Plasminogen Activator (TPA)(TPA)

TPA is used to treat coronary thrombosis TPA is used to treat coronary thrombosis (thrombolytic agent)(thrombolytic agent)

Protease that attaches to blood clots and induces other Protease that attaches to blood clots and induces other blood components to break down clotblood components to break down clot

**** Plasminogen Plasminogen Plasmin Plasmin

Fibrin ----------Fibrin ---------- Degradation of fibrin Degradation of fibrin

Streptokinase once used for this purposeStreptokinase once used for this purpose Derived from Streptococcal bacteriaDerived from Streptococcal bacteria Must be delivered to blood vessel directlyMust be delivered to blood vessel directly Urokinase = alternative, but has risk of Urokinase = alternative, but has risk of

hemorrhagehemorrhage

TPA does not compromise blood TPA does not compromise blood clotting elsewhere clotting elsewhere therefore therefore reduces risk of internal hemorrhagingreduces risk of internal hemorrhaging

Administered IV Administered IV transported via transported via circulation to affected areacirculation to affected area

Produced in mammalian cell culture Produced in mammalian cell culture (not E.coli)(not E.coli)

Early 1980s – Plasmid constructedEarly 1980s – Plasmid constructed Shuttle vector had characteristics for Shuttle vector had characteristics for

maintenance in E. coli and expression in maintenance in E. coli and expression in mammalian cellsmammalian cells

cDNA for TPAcDNA for TPA TPA signal sequenceTPA signal sequence TPA promoterTPA promoter TPA termination sitesTPA termination sites Antibiotic resistance + ORI for prokaryotic cellsAntibiotic resistance + ORI for prokaryotic cells Methotrexate resistance marker for mammalian cellsMethotrexate resistance marker for mammalian cells

Mammalian cells transfected Mammalian cells transfected Stable Stable integration into genome of mammalian host cellintegration into genome of mammalian host cell

High levels of secreted TPA produced in large High levels of secreted TPA produced in large scale culture conditionsscale culture conditions

FDA approves “Activase” – Genetech FDA approves “Activase” – Genetech – 1987– 1987 Clot-Dissolving AgentClot-Dissolving Agent Fewer side effects than streptokinase Fewer side effects than streptokinase

and urokinaseand urokinase

B. InterferonB. Interferon

and and are induced by “dsRNA” in are induced by “dsRNA” in cells infected by virusescells infected by viruses

Bind to neighboring cells and send a Bind to neighboring cells and send a warning signal of viruses nearby warning signal of viruses nearby (species-specific receptors)(species-specific receptors)

Neighboring cells protect themselves Neighboring cells protect themselves by producing proteins that inhibit viral by producing proteins that inhibit viral replicationreplication

IFNs also activate Natural Killer (NK) IFNs also activate Natural Killer (NK) cellscells

Before rDNA Before rDNA 90,000 pints of blood 90,000 pints of blood needed to purify 1 g IFNsneeded to purify 1 g IFNs

1980 – recombinant 1980 – recombinant -IFN -IFN Mammalian cell culture (glycosylated)Mammalian cell culture (glycosylated) Shown to:Shown to:

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

““Intron” marketed in 1984 by Swiss Biotech Intron” marketed in 1984 by Swiss Biotech FirmFirm

FDA approved its used in 1986 for use against FDA approved its used in 1986 for use against a particular form of leukemia, and in 1988 for a particular form of leukemia, and in 1988 for genital wartsgenital warts

Also used to treat Also used to treat Kaposi’s Sarcoma in AIDS patientsKaposi’s Sarcoma in AIDS patients Malignant melanomaMalignant melanoma Multiple myelomaMultiple myeloma Some kidney cancersSome kidney cancers

-IFN 1b licensed in 1993 for Multiple Sclerosis-IFN 1b licensed in 1993 for Multiple Sclerosis

IV. Other Innovative IV. Other Innovative PharmaceuticalsPharmaceuticals

A. Amgen ProductsA. Amgen Products

ErythropoietinErythropoietin Kidney-derived hormone that stimulates Kidney-derived hormone that stimulates

RBC production in the bone marrowRBC production in the bone marrow ““Epogen” – 1989Epogen” – 1989 Recombinant hormone used to alleviate Recombinant hormone used to alleviate

severe anemia that is a complication of severe anemia that is a complication of many kidney diseasesmany kidney diseases

““Neupogen”Neupogen” Stimulates stem cells to produce Stimulates stem cells to produce

neutrophils (and other leukocytes)neutrophils (and other leukocytes)

B. CytokinesB. Cytokines

Cytokines = hormone-like Cytokines = hormone-like substances that stimulate substances that stimulate lymphocyte (and some leukocyte) lymphocyte (and some leukocyte) activitiesactivities InterferonsInterferons Colony-stimulating factorsColony-stimulating factors Interleukin-2 (T cell growth factor)Interleukin-2 (T cell growth factor)

C.C. Monoclonal AntibodiesMonoclonal Antibodies

Specific antibodies produced Specific antibodies produced in vitroin vitro which can bind to:which can bind to: Cytokines (anticytokine Abs)Cytokines (anticytokine Abs) Specific subsets of cells Specific subsets of cells

Tumor cellsTumor cells MoAb against cell-surface tumor Ags can be used MoAb against cell-surface tumor Ags can be used

for diagnosis and immunotherapyfor diagnosis and immunotherapy MoAb can be conjugated to toxins or radioactive MoAb can be conjugated to toxins or radioactive

isotypes to kill tumor cells = Immunotoxinsisotypes to kill tumor cells = Immunotoxins Attacking T cells in graftsAttacking T cells in grafts

MoAbs can be “humanized” to MoAbs can be “humanized” to eliminate hypersensitivity reactions eliminate hypersensitivity reactions (serum sickness)(serum sickness) Replace “Fc” portion of MoAb with human Replace “Fc” portion of MoAb with human

“Fc” portion“Fc” portion Retain “Fab” – which contains the Retain “Fab” – which contains the

antigen-binding siteantigen-binding site rDNA techniques using cDNA can be rDNA techniques using cDNA can be

employed to cut and splice appropriate employed to cut and splice appropriate regions, followed by transfection into regions, followed by transfection into myrelomamyreloma

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 VaccinesV. DNA Vaccines

DNA vaccine technology is showing DNA vaccine technology is showing increasing promise in the treatment of increasing promise in the treatment of disease in humansdisease in humans

Numerous animal models are under Numerous animal models are under investigation for the use of DNA vaccines in investigation for the use of DNA vaccines in humanshumans MalariaMalaria AIDSAIDS HerpesHerpes TuberculosisTuberculosis Rotavirus (childhood diarrhea)Rotavirus (childhood diarrhea)

Differences between traditional Differences between traditional vaccines and DNA vaccinesvaccines and DNA vaccines Just the DNA coding for a specific Just the DNA coding for a specific

component of a disease-causing component of a disease-causing organism in injected into the body organism in injected into the body

Saline solution/hypodermic needleSaline solution/hypodermic needle DNA-coated gold beads propelled into the DNA-coated gold beads propelled into the

body using gene guns body using gene guns

Production of the immunizing protein occurs Production of the immunizing protein occurs in the vaccinated hostin the vaccinated host

Once inside cells, some of the rDNA go to Once inside cells, some of the rDNA go to the nucleus and instruct transcription of the nucleus and instruct transcription of encoded antigenic proteinsencoded antigenic proteins

Protein products can elicit humoral (Ab-type) IR if Protein products can elicit humoral (Ab-type) IR if the proteins are released from the cells, or cell-the proteins are released from the cells, or cell-mediated (killer-type) immunity is protein are mediated (killer-type) immunity is protein are processed/degraded intracellulary and properly processed/degraded intracellulary and properly displayed on the cell surfacedisplayed on the cell surface

Which cells take up rDNA is critically important for Which cells take up rDNA is critically important for efficient recognition of antigen molecules efficient recognition of antigen molecules (professional antigen presenting cells required for (professional antigen presenting cells required for efficient T cell responses)efficient T cell responses)

Risk of infection associated with live or Risk of infection associated with live or attenuated virus vaccines is eliminatedattenuated virus vaccines is eliminated

DNA vaccination provides long-lived DNA vaccination provides long-lived immune response (boosters not need to immune response (boosters not need to maintain immunity)maintain immunity)

Vaccines can be produced using similar Vaccines can be produced using similar techniques (simplification of techniques (simplification of development and production processes)development and production processes)

Stable (dried or in solution)Stable (dried or in solution)

As microbial genomes of pathogens are As microbial genomes of pathogens are sequenced, the sequence information sequenced, the sequence information can be used for vaccine design can be used for vaccine design

Potential Risks:Potential Risks: Potential for induction for tolerancePotential for induction for tolerance Random integration into the genome?Random integration into the genome? Are plasmid vectors toxic? Does DNA Are plasmid vectors toxic? Does DNA

delivered as a drug incite an immune delivered as a drug incite an immune response against the body’s own DNA?response against the body’s own DNA?