THERAPEUTIC AGENTS N7-2006 L. Duroux Slides assembled from diverse sources.

THERAPEUTIC THERAPEUTIC AGENTSAGENTS

N7-2006N7-2006L. DurouxL. Duroux

Slides assembled from diverse sourcesSlides assembled from diverse sources

Recommended reading list - textbooksRecommended reading list - textbooks

Novel Therapeutic Proteins (2001)Peter Stadler & Klaus DembowskyPeter Stadler & Klaus Dembowsky Wiley-VCH, Wiley-VCH, ISBN ISBN 35273027003527302700

Human Gene Therapy (2003)Gabor M. Rubanyi & Seppo Ylä-HerttualaGabor M. Rubanyi & Seppo Ylä-Herttuala

Springer, ISBN ISBN 35400041303540004130

The Development of Human Gene Therapy (1999)Theodore FriedmannTheodore Friedmann

CSHL Press, ISBN 0879695285CSHL Press, ISBN 0879695285

Lecture PlanLecture Plan

1.1. IntroductionIntroduction

2.2. Recombinant ProteinsRecombinant Proteins

3.3. Nucleic AcidsNucleic Acids

1. INTRODUCTION1. INTRODUCTION

Therapeutic AgentsTherapeutic Agents

Before the advent of molecular Before the advent of molecular biotechnology most biotechnology most human proteinshuman proteins were available in only were available in only small small (limited) (limited) quantities.quantities.

Today hundreds of genes (~1000) for Today hundreds of genes (~1000) for human proteins have been cloned, human proteins have been cloned, sequenced, expressed in the host sequenced, expressed in the host cells and are being tested as cells and are being tested as therapeutic agents (drugs) in humans.therapeutic agents (drugs) in humans.

Biotechnology TodayBiotechnology Today 4400 biotech companies worldwide4400 biotech companies worldwide

Sales of $40-50 billion Sales of $40-50 billion

1500-2000 biotech companies in US1500-2000 biotech companies in US 160,000 employees160,000 employees

Major biotech companies include Genetech, Major biotech companies include Genetech, Amgen, BiogenIdec, Genzyme, Centocor, Amgen, BiogenIdec, Genzyme, Centocor, MedImmune, Chiron, Novo Nordisk, SeronoMedImmune, Chiron, Novo Nordisk, Serono

Many large pharma companies investing in Many large pharma companies investing in biotechbiotech

A. Demain, 2004, ChimicaOggi

BiopharmaceuticalsBiopharmaceuticals Over 140 on the market; over 400 in clinical trialsOver 140 on the market; over 400 in clinical trials

Biopharmaceuticals include:Biopharmaceuticals include: Proteins (made in bacterial, fungal or mammalian cell culture)Proteins (made in bacterial, fungal or mammalian cell culture)

• erythropoietin (EPO) erythropoietin (EPO) • insulin insulin • interferon (Intron A)interferon (Intron A)• granulocyte-colony stimulating factor (G-CSF)granulocyte-colony stimulating factor (G-CSF)• human growth hormone (HGH, human somatotropin)human growth hormone (HGH, human somatotropin)• tissue plasminogen activator (tPA) tissue plasminogen activator (tPA)

Monoclonal antibodies (made in mammalian cell culture)Monoclonal antibodies (made in mammalian cell culture) VaccinesVaccines

• live and inactivated viruses and bacterialive and inactivated viruses and bacteria• subunit vaccinessubunit vaccines• recombinant vaccinesrecombinant vaccines

Gene Therapy Products (viral and non-viral)Gene Therapy Products (viral and non-viral)

Product

Manufacturer Launch I ndication

OKT3 J &J 1986 Transplant rejection Murine

ReoPro Eli Lilly 1994 Cardiovascular disease Chimeric Rituxan I dec 1997 Non-Hodgkin’s lymphoma Chimeric Zenapax Roche 1997 Organ rejection Humanized

Simulect Novartis 1998 Organ rejection Chimeric Remicade Centocor 1998 Rheumatoid arthritis Chimeric Herceptin Genentech 1998 Breast cancer Humanized

Synagis MedI mmune 1998 Respiratory syncytial virus Humanized Enbrel I mmunex 1998 Rheumatoid arthritis Human

Mylotarg Wyeth 2000 Leukemia Humanized Campath Millennium 2001 Lymphocytic leukaemia Humanized Zevalin I dec 2002 Non-Hodgkin’s lymphoma Murine

Humira Abbot 2002 Rheumatoid arthritis Human Xolair Genentech 2003 Asthma Humanized

Bexxar Corixa/GSK 2003 Non-Hodgkin’s lymphoma Murine Raptiva Xoma/Genen. 2003 Psoriasis Humanized Erbitux I mclone 2004 Cancer (colorectal) Chimeric

Avastin Genen. 2004 Cancer (colon or rectum) Humanized

FDA-Approved AntibodiesFDA-Approved Antibodies

Recombinant Protein ProductionRecombinant Protein Production(including MAbs)(including MAbs)

Cells and plasmid

+

Cell line

Transfection Cell culture Purification

Drug substance (crude)

Drug substance (pure)

Drug product -(sterile)

Formulation/Filling

Bioprocess Engineering in Bioprocess Engineering in Recombinant Protein ProductionRecombinant Protein Production

Cells and plasmid

+

Cell line

Transfection Cell culture Purification

Drug substance (crude)

Drug substance (pure)

Drug product -(sterile)

Formulation/Filling

Cell line manufacture

Medium development

Bioreactor process development & scale-up

Downstream purification

Analytical characterization

BIOPROCESS ENGINEERINGBIOPROCESS ENGINEERING Natural productsNatural products

mostly from fungi and actinomyctesmostly from fungi and actinomyctes filamentous organismsfilamentous organisms many anti-fungal, anti-bacterial, anti-many anti-fungal, anti-bacterial, anti-

cancer compoundscancer compounds also statins for lowering cholesterol are also statins for lowering cholesterol are

natural products and semi-synthetics: natural products and semi-synthetics: Zocor (Merck), Lipitor (Pfizer)Zocor (Merck), Lipitor (Pfizer)

BIOPROCESS ENGINEERINGBIOPROCESS ENGINEERING Gene Therapy ProductsGene Therapy Products

gene delivered by one of many vehiclesgene delivered by one of many vehicles Non-viral vectors: naked DNA, DNA Non-viral vectors: naked DNA, DNA

plasmid, liposomesplasmid, liposomes Viral vectors: adenovirus, retrovirus, Viral vectors: adenovirus, retrovirus,

lentivirus, adeno-associated viruslentivirus, adeno-associated virus

J.R. Morgan and M.L. Yarmush, Gene Therapy in Tissue Engineering. In:C.W. Patrick, A.G. Mikos, L.V. McIntire, eds. Frontiers in Tissue Engineering, Elsevier Science Publishers, 1997

The Drug Development ProcessThe Drug Development Process

Drug Discovery

Lead Optimization(metabolism,toxicology)

ProcessDevelopment/Material Generation

Animal &ClinicalTesting

Iterative Process

Regulatory Considerations

Bioprocess DevelopmentBioprocess Development

Cell Line/Viral Vector/Construction

Cell/VirusCultureDevelopment/Media Optimization

PurificationProcess Dev.

FormulationDesign/Drug DeliveryDesign

Biological Assay Development and Support

Facility/Equipment DesignTechnology TransferProcess ValidationPilot-Scale cGMP ProductionCommercial-Scale Production

2. Recombinant 2. Recombinant ProteinsProteins

2A. Human Interferons2A. Human Interferons

Fighting Viral InfectionsFighting Viral Infections

Mechanisms of Resistance Mechanisms of Resistance against Infectionsagainst Infections

What is Interferon?What is Interferon?

Scientists discovered an antiviral protein Scientists discovered an antiviral protein in 1957 that inhibited growth of influenza in 1957 that inhibited growth of influenza virus in chicken embryos. It was named virus in chicken embryos. It was named interferon because it interfered with the interferon because it interfered with the growth of influenza virus.growth of influenza virus.

InterferonsInterferons

Anti viral proteins released by host cellsAnti viral proteins released by host cells Interfere with viral multiplicationInterfere with viral multiplication

Host cell specific but not virus specificHost cell specific but not virus specific

Different types of cells in animals produce Different types of cells in animals produce different interferonsdifferent interferons

Human InterferonHuman Interferon

3 types3 types alpha interferon (13 genes)alpha interferon (13 genes) beta interferon (2 genes)beta interferon (2 genes) gamma interferon (1 gene)gamma interferon (1 gene)

Alpha & beta usually produced early in viral Alpha & beta usually produced early in viral infections (viruses or viral RNA)infections (viruses or viral RNA)

Gamma appears laterGamma appears later

InterferonInterferon

Presence of double-stranded RNA Presence of double-stranded RNA indicates cell is infectedindicates cell is infected

Viral infected cells release alpha and beta Viral infected cells release alpha and beta

interferonsinterferons Diffuse to neighboring cellsDiffuse to neighboring cells

Virus can’t replicateVirus can’t replicate

Antiviral TreatmentAntiviral Treatment

Interferon therapyInterferon therapy Limited lifetime, short lasting effectLimited lifetime, short lasting effect

Recombinant interferonsRecombinant interferons• Pure and fastPure and fast• Hybrid genes for enhanced/new activityHybrid genes for enhanced/new activity

Oral administrationOral administration

AMARILLO Biosciences AMARILLO Biosciences Inc.’s IFNInc.’s IFN

Amarillo Biosciences, Inc. (ABI)Amarillo Biosciences, Inc. (ABI)(OTCBB:AMAR)(OTCBB:AMAR)

806-376-1741 806-376-1741 Fax: 806-376-9301 Fax: 806-376-9301

E-mail: E-mail: [email protected]@amarbio.com

October 31, 2006October 31, 2006

The CompanyThe Company

Amarillo Cell Culture Company, Inc. was formed in 1984. The Company is the world leader in the development of low-dose

interferon-alpha for oral delivery to humans and animals. Before an initial public offering in 1996, the name of the

Company was changed to Amarillo Biosciences, Inc. (ABI). The Company’s goal is to achieve regulatory approval of a

safe and effective low dose oral formulation of a human interferon alpha.

Company OverviewCompany Overview• Founded in 1984

• Product focus - oral delivery of interferon alpha:

8 active Investigational New Drug (IND) applications in USA

24 human clinical trials completed or ongoing

50 animal clinical trials worldwide

• Advanced clinical development programs

Phase 2 in oral warts for HIV+ patients – enrollment starting in November

Phase 2 in Behcet’s disease in Turkey – 39 of 90 patients enrolled

Phase 2 in chronic cough planned

• Strategic alliance with Hayashibara Biochemical Laboratories

Injectable interferon is approved world-wide for the Injectable interferon is approved world-wide for the

treatment of various cancers and viral diseases.treatment of various cancers and viral diseases.

Interferon is a protein readily eliminated from the blood Interferon is a protein readily eliminated from the blood

by the kidney. To counteract the kidney’s clearance of by the kidney. To counteract the kidney’s clearance of

interferon from the blood injectable interferon must be interferon from the blood injectable interferon must be

given in doses much higher than what occur naturally.given in doses much higher than what occur naturally.

Why are Side Effects Common and Why are Side Effects Common and Severe Severe

for Injectable Interferon?for Injectable Interferon?

How is Oral Interferon Different?How is Oral Interferon Different?

Low-dose oral interferon is given in doses 10 thousand Low-dose oral interferon is given in doses 10 thousand times less than injectable interferon. Therefore, side times less than injectable interferon. Therefore, side effects are dramatically reduced.effects are dramatically reduced.

Oral interferon is human interferon alpha administered in a Oral interferon is human interferon alpha administered in a small tablet (lozenge) to humans or in powder to animals.small tablet (lozenge) to humans or in powder to animals.

Oral interferon binds to surface (mucosal) cells in the Oral interferon binds to surface (mucosal) cells in the mouth and throat resulting in stimulation of white blood mouth and throat resulting in stimulation of white blood cells and activates hundreds of genes affecting the cells and activates hundreds of genes affecting the immune system in the peripheral blood of man, cattle and immune system in the peripheral blood of man, cattle and mice.mice.

Studies show oral interferon is effective against disorders Studies show oral interferon is effective against disorders such as cancer, viral diseases and autoimmunity. such as cancer, viral diseases and autoimmunity.

What is Special about ABI’s Oral What is Special about ABI’s Oral Interferon?Interferon?

Comparison Oral Injectable

Dose Up to 500 IU Up to 10,000,000 IU

Side Effects Rare/mild Common/severe

Treatment Cost Cost Effective Expensive

Administration Lozenge/powder Needle/syringe

Physician Visits Infrequent Frequent

Stability Stable at Room Refrigeration Required

Temperature

Unique Oral Formulation

VELDONA (VVery Low Dose Oral Natural Alpha) is the brand name of the interferon lozenges which have been tested in thousands of patients. The formula is stable at room temperature for 2 years and at refrigeration temperature for 5 years.

The combination of IFN subspecies (INF2b, INF7 and INF8) provide superior immunomodulatory,

anticancer and antiviral effects.

Manufacturing Steps for ABI’s Interferon

Mechanism of ActionMechanism of Action

Interferon placed in the mouth binds to receptors in the mucosal lining and initiates systemic effects on the immune system in animals and man. These immunomodulatory effects are safe and effective in helping control viral and autoimmune diseases and cancer.

IFN

Oral Epithelial Cells

Tonsil

Mandibular Lymph Nodes

Activation of Cell Mediated

ImmunityVirus

Activation of Humoral Immunity (Antibody)

Activation of Perioral Lymphoid Cells and Peripheral Lymphoid Tissues

The Company has 13 issued and one pending patent with claims to the manufacture of the oral formulation or the use of interferon orally to manage numerous diseases. ABI has clinical data on safety and efficacy from 24 human and 50 animal studies.

Intellectual Property

ABI’s Pipe Line for Human HealthABI’s Pipe Line for Human Health

Behcet's disease Chronic active hepatitis BSjogren's syndrome Chronic hepatitis CIdiopathic pulmonary fibrosis Wasting cancer patientsMultiple sclerosis FibromyalgiaAphthous stomatitis MeaslesOral mucositis - cancer Respiratory syncytial virusLichen planus InfluenzaOpportunistic infections - HIV+ Cough (COPD & IPF)

Human diseases in which oral interferon has been tested and reported to be safe

The market for Injectable interferon exceeds $5 billion annually.

The Company believes low dose oral interferon is superior to injectable in safety, cost and ease of administration. The Company is working toward FDA approval for oral interferon to treat influenza, Behcets disease, oral warts in HIV+ patients and in idiopathic pulmonary fibrosis. Once oral interferon is FDA approved, the Company projects sales to exceed $100 million annually.

Oral Interferon Human Health Market Europe and United States

DiseaseDisease PopulationPopulation Potential Potential MarketMarket

Oral Warts in HIV+ Oral Warts in HIV+ PatientsPatients

80,00080,000 $170 million$170 million

Behcet’s DiseaseBehcet’s Disease 39,00039,000 $40 million$40 million

Influenza (seasonal)Influenza (seasonal)

Cough (COPD & IPF)Cough (COPD & IPF)

77 million77 million

39 million39 million

Over $1 billionOver $1 billion

Over $1 billionOver $1 billion

Influenza - IntroductionInfluenza - Introduction Highly contagious, acute respiratory illness known Highly contagious, acute respiratory illness known

as influenza has caused epidemics and pandemics as influenza has caused epidemics and pandemics of humans and animals for centuries. of humans and animals for centuries.

About 20% of children and 5% of adults worldwide About 20% of children and 5% of adults worldwide develop symptomatic influenza each year.develop symptomatic influenza each year.

Most influenza infections are spread by virus–laden Most influenza infections are spread by virus–laden respiratory droplets through coughing and sneezing.respiratory droplets through coughing and sneezing.

Influenza can be transmitted to people by pigs or Influenza can be transmitted to people by pigs or birds.birds.

• 14,000 people participated in controlled studies of placebo versus interferon treatment during a natural outbreak of Hong Kong influenza.

• Interferon (about 128 units) or placebo was dripped into the nose daily for 5 days starting about the time of the first reported influenza cases.

• Interferon significantly (P<0.01) reduced the number of influenza cases.

Human Study – Influenza and Interferon

Soloviev, Bull. WHO 41:683-688, 1969.

Efficacy of Human Leukocyte Interferon as Prophylaxis Against Influenza

0

5

10

15

20

25

Adults Children7-12 yr

Children2-6 yr

Population (14,000 subjects total)

Per

cen

t o

f P

atie

nts

Sic

k

Interferon

Placebo

Oral Interferon Protected Mice Against a Fatal Challenge of Influenza

14

14.5

15

15.5

16

16.5

17

17.5

18

18.5

19

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

days post infection

weig

ht

(g)

0 IU IFN/dose

1 IU IFN/dose

10 IU IFN/dose

100 IU/dose

p < 0.489

p < 0.889

p < 0.0002

*

Summary - Oral Interferon Summary - Oral Interferon

Patented oral interferon technology upregulates hundreds of Patented oral interferon technology upregulates hundreds of genes affecting immune function - stable at room temperature genes affecting immune function - stable at room temperature for 2 years, safe and effective without significant side effects, for 2 years, safe and effective without significant side effects, easy to administer.easy to administer.

Injectable interferon market is $5 billion for treatment of Injectable interferon market is $5 billion for treatment of cancer, autoimmune and viral diseases although side effects cancer, autoimmune and viral diseases although side effects are moderate to severe for many patients. are moderate to severe for many patients.

Potential US & European markets for oral interferon treatment Potential US & European markets for oral interferon treatment of oral warts in HIV+ patients, Behcet’s disease, influenza and of oral warts in HIV+ patients, Behcet’s disease, influenza and cough – over $1 billioncough – over $1 billion

Company Goal - Achieve regulatory approval of a safe and Company Goal - Achieve regulatory approval of a safe and effective low dose oral formulation of a human interferon effective low dose oral formulation of a human interferon alpha.alpha.

Strategies for Optimisation of Strategies for Optimisation of Recombinant ProductionRecombinant Production

Screening libraries of recombinant genes Screening libraries of recombinant genes (IFNs, human growth hormone, TNF-a…)(IFNs, human growth hormone, TNF-a…)

Screening of recombinant expression Screening of recombinant expression systems (E coli, fungi, Mammalian cells…)systems (E coli, fungi, Mammalian cells…)

Delivery by intestinal bacteria (lactobacilli)Delivery by intestinal bacteria (lactobacilli)

2B. Enzymes2B. Enzymes

Treating Cystic FibrosisTreating Cystic Fibrosis

A Fatal Genetic DiseaseA Fatal Genetic Disease

Cystic fibrosis (CF) is one of the most fatal heredity Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with diseases among European and their descendants with ~30,000 cases in the US and 23,000 in Canada.~30,000 cases in the US and 23,000 in Canada.

Furthermore among European descendants it occurs in 1 in Furthermore among European descendants it occurs in 1 in 2,500 live birth and 1 in 25 are carriers.2,500 live birth and 1 in 25 are carriers.

It is caused by more than 500 different mutations in the It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator cystic fibrosis transmembrane conductance regulator (CFTR) gene.(CFTR) gene.

Individuals with CF are highly susceptible to bacterial Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant infection and antibiotic treatment often results in resistant strains.strains.

Symptoms of Cystic FibrosisSymptoms of Cystic Fibrosis

Clogging and infection of lungsClogging and infection of lungs plugging of small bile ducts in liver (impedes plugging of small bile ducts in liver (impedes

digestion)digestion) plugging of ducts of pancreas (impedes plugging of ducts of pancreas (impedes

digestion)digestion) obstruction of small intestineobstruction of small intestine males are infertile (blocked vas deferens)males are infertile (blocked vas deferens) malfunctioning sweat glandsmalfunctioning sweat glands

The theme is that small ducts become clogged with a thick mucus

A variety of A variety of organs are organs are

affectedaffected

A large effort was launched to A large effort was launched to identify the gene responsibleidentify the gene responsible

Identified a gene on chromosome 7Identified a gene on chromosome 7

CF was caused by a 3 base pair CF was caused by a 3 base pair deletion in Cystic Fibrosis deletion in Cystic Fibrosis Transmembrane Regulator (CTFR)Transmembrane Regulator (CTFR)

The gene is very largeThe gene is very large

The CF geneThe CF gene

How could such a minor change How could such a minor change cause such a dramatic effect?cause such a dramatic effect?

F508 is deletedF508 is deleted The encoded protein looks like an ABC The encoded protein looks like an ABC

transporter coupled to a channeltransporter coupled to a channel If the mutant protein is isolated and put If the mutant protein is isolated and put

into a membrane it will function normallyinto a membrane it will function normally So what is wrong?So what is wrong?

Improperly folded proteins are not Improperly folded proteins are not allowed to leave the ERallowed to leave the ER

Currently theories are that the protein is not recognized as being properly folded

How does this protein work?How does this protein work?

The gene is a pore coupled to two ABCsThe gene is a pore coupled to two ABCs It transports Cl- ions after being It transports Cl- ions after being

phosphorylated and binding two ATP phosphorylated and binding two ATP moleculesmolecules

It has a large regulatory domain that is It has a large regulatory domain that is phosphorylated by a cAMP dependent phosphorylated by a cAMP dependent protein kinaseprotein kinase

The protein’s physiological role

A normal lungChloride into airway; sodium out - keeps mucus moist and thinNormal CFTR regulates the sodium channel (inactivates it)

A CF LungA CF Lung

Chloride does not get into airway; more sodium leaves; More salt in cell - water comes inThis makes the mucus thick

The result:The result:physiology of a normal lungphysiology of a normal lung

The result: The result: physiology of a CF lungphysiology of a CF lung

TreatmentsTreatments

Chloride delivery - Chloride delivery - activate other chloride activate other chloride carrierscarriers

Viscous mucus - Viscous mucus - pounding, DNase pounding, DNase treatment, gelosintreatment, gelosin

recurrent infections - recurrent infections - antibioticsantibiotics tissue damage due to immune response - tissue damage due to immune response -

anti-inflammatory drugs (ibuprofen)anti-inflammatory drugs (ibuprofen)

DNase 1 (GeneTech)DNase 1 (GeneTech)

A thick mucus which is a results of:A thick mucus which is a results of: Alignate produced by bacteriaAlignate produced by bacteria DNA from lysed cellsDNA from lysed cells Leucocytes which accumulate due to the infectionLeucocytes which accumulate due to the infection

Makes breathing difficult.Makes breathing difficult. Scientist at Genentech isolated the gene for Scientist at Genentech isolated the gene for

DNase1DNase1 The purified enzyme was delivered as an aerosol The purified enzyme was delivered as an aerosol

to the lung where it hydrolysed the DNA into short to the lung where it hydrolysed the DNA into short oligonucleotides.oligonucleotides.

This decrease the viscosity in the lungs and made This decrease the viscosity in the lungs and made breathing easier.breathing easier.

Alginate LyaseAlginate Lyase Alginate is a polysaccharide polymer that is Alginate is a polysaccharide polymer that is

produced by seaweed and some soil and produced by seaweed and some soil and marine bacteria.marine bacteria.

The excretion of alginate by The excretion of alginate by Pseudomonas Pseudomonas aeruginosaaeruginosa of patients with CF contributes of patients with CF contributes to the viscosity in the lung.to the viscosity in the lung.

The enzyme alginate lyase can liquefy The enzyme alginate lyase can liquefy bacteria alginate.bacteria alginate.

Alginate lyase was isolate from Alginate lyase was isolate from Flavobacterium Flavobacterium sp. and cloned into sp. and cloned into E. coliE. coli..

Alginate LyaseAlginate Lyase

The expressed gene produced a protein of The expressed gene produced a protein of 69,000 Da.69,000 Da.

The 69,000 Da protein produced a The 69,000 Da protein produced a proteolytic enzyme of 6,000 Da.proteolytic enzyme of 6,000 Da.

The remain 63,000 Da protein was cleaved The remain 63,000 Da protein was cleaved to produce a 43,000 Da which is able to to produce a 43,000 Da which is able to liquefy bacterial alginate.liquefy bacterial alginate.

Combined with DNase1, alginate lyse is Combined with DNase1, alginate lyse is able to reduce the mucus in the lungs of able to reduce the mucus in the lungs of patients with CF.patients with CF.

Alginate lyaseAlginate lyase

2C. Monoclonal 2C. Monoclonal AntibodiesAntibodies

Clinical ApplicationsClinical Applications Transplantation – muronomab (OKT3) 1986, Transplantation – muronomab (OKT3) 1986,

basiliximab 1998basiliximab 1998

Cardiovascular disease – abciximab 1994Cardiovascular disease – abciximab 1994

Cancer – rituximab 1997, trastuzumab 1998Cancer – rituximab 1997, trastuzumab 1998

Viral infection – palivizumab 1998Viral infection – palivizumab 1998

Inflammatory diseases – infliximab 1998, Inflammatory diseases – infliximab 1998, etanercept 1999etanercept 1999

Side effectsSide effects

Transfusion reactionsTransfusion reactions

Infections, immunosuppressionInfections, immunosuppression

Cardiac, respiratory arrestCardiac, respiratory arrest

Pharmacological toxicityPharmacological toxicity

Production of Monoclonal AntibodiesProduction of Monoclonal Antibodies

Monoclonal antibodies results from a clone Monoclonal antibodies results from a clone of a of a B lymphocyteB lymphocyte producing a single producing a single antibody which will bind to a specific antibody which will bind to a specific epitopeepitope of an antigen. of an antigen.

What is a polyclonal antibody?What is a polyclonal antibody?

Monoclonal antibodies are produced:Monoclonal antibodies are produced: Fusion of a Fusion of a myeloma myeloma (B cell which has (B cell which has

become cancerous) with a spleen cell that is become cancerous) with a spleen cell that is immunized with a specific antigen.immunized with a specific antigen.

The resulting The resulting hybridomashybridomas are tested for are tested for the production of a monoclonal antibodies.the production of a monoclonal antibodies.

Background and developmentBackground and development

Diluted to one cell culturesDiluted to one cell cultures Hypoxanthine Guanine Phosphoribosyl Hypoxanthine Guanine Phosphoribosyl

Transferase (HGPT) negative myeloma Transferase (HGPT) negative myeloma cellscells

Grown in Hypoxanthine Aminopterin Grown in Hypoxanthine Aminopterin Thymidine (HAT) mediumThymidine (HAT) medium

De novo nucleotide synthesis blockedDe novo nucleotide synthesis blocked Only successful fusion cells grow (rare)Only successful fusion cells grow (rare)

Production Production of of

Monoclonal Monoclonal AntibodiesAntibodies

Major Problems with non-human ABs:

Immunological Responses

”Humanization” of ABs

The Structure of AntibodiesThe Structure of Antibodies

65 – 90% human and consist of the murine variable regions ( fused to the constant or effector part)

Chimeric Ab

Production of Human Monoclonal Production of Human Monoclonal Antibodies by Antibodies by E. coliE. coli

Hybridoma cells grow relatively slow and require Hybridoma cells grow relatively slow and require expensive media.expensive media.

To circumvent this problem human monoclonal To circumvent this problem human monoclonal antibodies are grown in antibodies are grown in E. coliE. coli..

The produce involves:The produce involves: mRNA mRNA is isolated from the B cell.is isolated from the B cell. cDNAcDNA is synthesized from the mRNA by the is synthesized from the mRNA by the

enzyme enzyme reverse transciptasereverse transciptase.. Both Both heavy and light chainsheavy and light chains are amplified are amplified

separately from the cDNA using PCR.separately from the cDNA using PCR. The amplified products are cut with restriction The amplified products are cut with restriction

enzymes and enzymes and cloned into Lambda vectorcloned into Lambda vector..

Production Production of Human of Human

Monoclonal Monoclonal Antibodies Antibodies by by E. coliE. coli

Production of Human Monoclonal Production of Human Monoclonal Antibodies by Antibodies by E. coliE. coli

During cloning different light and heavy chains During cloning different light and heavy chains are cloned.are cloned.

The DNA of one heavy and one light chain are The DNA of one heavy and one light chain are cloned into the same vector.cloned into the same vector.

Many Many different combinationsdifferent combinations of H and L chains of H and L chains are cloned together in the same vector.are cloned together in the same vector.

Lambda is not useful for producing large Lambda is not useful for producing large amounts of proteins.amounts of proteins.

The L and H chains are excised from Lambda The L and H chains are excised from Lambda and cloned into an and cloned into an E. coliE. coli plasmid and the plasmid and the recombinant plasmid transformed into recombinant plasmid transformed into E. coliE. coli..

Production Production of Human of Human

Monoclonal Monoclonal Antibodies Antibodies by by E. coliE. coli

95% human, and are made by grafting the hypervariable region (or CDR) of the murine antibody –which determines Ab specificity

Humanized Ab

Isolation of CDR SegmentsIsolation of CDR Segments

PCR amplification of CDR segments from PCR amplification of CDR segments from mouse monoclonal antibody to desired targetmouse monoclonal antibody to desired target

Insert into cDNA clone of human antibodyInsert into cDNA clone of human antibody Produce human antibody with desired CDRs Produce human antibody with desired CDRs

and antigen specificityand antigen specificity

Combinatorial LibraryCombinatorial Library

Create new Create new variations of variations of antibodies antibodies New combinations of New combinations of

heavy and light chainsheavy and light chains

mRNA from mRNA from immunized individualimmunized individual

PCR H and L chainsPCR H and L chains Clone into vector in Clone into vector in

new H/L new H/L combinationscombinations

Vector for Combinatorial LibraryVector for Combinatorial Library

Antibody Display LibrariesAntibody Display Libraries

Screening a M13 Combinatorial LibraryScreening a M13 Combinatorial Library

only one heavy-chain variable domain and one light-chain variable domain covalently linked by peptide

Single-chain Fixed variable

Single-Chain Combinatorial Antibody Library

Application : ImmunotoxinsApplication : Immunotoxins

Protein toxin connected to Fv regionProtein toxin connected to Fv region Single-chain or S-S linked Fv regionSingle-chain or S-S linked Fv region Toxin localized to antigen-expressing cellsToxin localized to antigen-expressing cells

2D. Examples of Ab 2D. Examples of Ab therapytherapy

Improved Drug Targeting Improved Drug Targeting

Targeting Thrombolytic Targeting Thrombolytic Plasminogen ActivatorPlasminogen Activator

Targeting TPATargeting TPA

‘In the absence of vascularisation, solid tumours remain dormant and 2–3mm3 in size, with size being limited by the ability of oxygen and nutrients to diffuse into the tumour’

Dr. Judah Folkman hypothesis (1971)

Folkman J. N Engl J Med 1971;285:1182–6

Tumor hypoxia model

180um

Hypoxic cell

vessels

TumorMass

Bergers G, et al. Nature 2002;3:401–10

Small tumour (1–2mm)• Avascular• Dormant

Larger tumour• Vascular• Metastatic potential

Angiogenic switch

• Recombinant humanised monoclonal antibody targeting the angiogenic

factor VEGF

• Similar to Herceptin®

: 93% human, 7% murine

Bevacizumab (Avastin®)

3. Nucleic Acids3. Nucleic Acids

3A. Methods3A. Methods

Nucleic Acids as Therapeutic agentsNucleic Acids as Therapeutic agents

Many human disorders e.g. cancer and Many human disorders e.g. cancer and inflammatory conditions (virus, parasites) are often inflammatory conditions (virus, parasites) are often caused by overproduction of a normal protein.caused by overproduction of a normal protein.

Theoretically a small ss nucleic acid can hybridize Theoretically a small ss nucleic acid can hybridize to a specific gene or mRNA and diminish to a specific gene or mRNA and diminish transcription or translation.transcription or translation.

An oligonucleotide (oligo) that binds to a gene and An oligonucleotide (oligo) that binds to a gene and blocks transcription is an antigene.blocks transcription is an antigene.

An oligo that binds to mRNA and blocks An oligo that binds to mRNA and blocks translation is called an antisense oligo.translation is called an antisense oligo.

Ribozyme (catalytic RNA) and interfering RNA Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific mRNA for degradation.( RNAi) can target specific mRNA for degradation.

Antisense RNAsAntisense RNAs

Gene segment Gene segment cloned into cloned into expression expression vector in reverse vector in reverse orientationorientation

Formation of Formation of dsRNA can dsRNA can interfere with interfere with RNA processing RNA processing or translationor translation

Antisense RNA : ApplicationsAntisense RNA : Applications

Episomally based expression vectors with cDNA Episomally based expression vectors with cDNA for insulin-like growth factor 1 (ILGF-1) receptors for insulin-like growth factor 1 (ILGF-1) receptors were constructed in the antisense version.were constructed in the antisense version.

ILGF-1 is prevalent in malignant glioma a common ILGF-1 is prevalent in malignant glioma a common form of brain cancer and prostate carcinoma.form of brain cancer and prostate carcinoma.

Culture of glioma cells when transfected with the Culture of glioma cells when transfected with the antisense version of ILGF-1 in ZnSOantisense version of ILGF-1 in ZnSO4 4 lost its lost its

tumurous properties.tumurous properties. A similar treatment of mice which were injected A similar treatment of mice which were injected

with prostate carcinoma cells caused small or no with prostate carcinoma cells caused small or no tumor to develop.tumor to develop.

Antisense OligonucleotidesAntisense Oligonucleotides

Antisense deoxynucleotides can also be Antisense deoxynucleotides can also be used as therapeutic agents. used as therapeutic agents.

However when injected into the body is However when injected into the body is deoxynucleotides are susceptible to deoxynucleotides are susceptible to degradation.degradation.

To prevent this modified deoxynucleotides To prevent this modified deoxynucleotides are used including phosphorothioate, are used including phosphorothioate, phosphoramidate and polyamide.phosphoramidate and polyamide.

Free oligos are usually introduced into to Free oligos are usually introduced into to the body encapsulated in a liposome.the body encapsulated in a liposome.

Modified DeoxynucleotidesModified Deoxynucleotides

Phosphodiester linkage Phosphorothioate linkage

Modified DeoxynucleotidesModified Deoxynucleotides

Phosphoramidite linkage Polyamide linkage

Liposome Delivery of Therapeutic Liposome Delivery of Therapeutic Nucleic AcidsNucleic Acids

Small vesicles of Small vesicles of phospholipid bilayerphospholipid bilayer

Fuse to cells and release Fuse to cells and release contents into cytoplasmcontents into cytoplasm

Approach can target human Approach can target human cells and/or infectious agent cells and/or infectious agent in early stage tests (e.g. in early stage tests (e.g. mycobacterium mycobacterium tuberculosistuberculosis))

Antisense Oligos and Antisense Oligos and PsoriasisPsoriasis

Antisense oligos have also been tested in the Antisense oligos have also been tested in the treatment of psoriasis.treatment of psoriasis.

Psoriasis is uncontrollable epidermal growth.Psoriasis is uncontrollable epidermal growth. ILGF-1 receptors are implicated in the ILGF-1 receptors are implicated in the

pathogenesis of psoriasis.pathogenesis of psoriasis. 15 nt antisense oligo were transferred into 15 nt antisense oligo were transferred into

keratinocytes using liposome and the amount of keratinocytes using liposome and the amount of ILGF-1 protein was decreased by 45-65%.ILGF-1 protein was decreased by 45-65%.

When mouse with human psoriasis lesions were When mouse with human psoriasis lesions were injected with anitsense oligi complementary to injected with anitsense oligi complementary to ILGF-1 receptor mRNA there was significant ILGF-1 receptor mRNA there was significant reduction (58-69%) in epidermal thickness.reduction (58-69%) in epidermal thickness.

Correct Mutation in Splice Site in Correct Mutation in Splice Site in beta-globin gene (Thalassemia)beta-globin gene (Thalassemia)

Synthetic Synthetic oligonucleotide blocks oligonucleotide blocks site of mutation which site of mutation which created mutant created mutant “alternative” splice “alternative” splice junctionjunction Acts at RNA levelActs at RNA level

RNAiRNAi RNA interference or RNA interference or

gene silencinggene silencing dsRNA processed dsRNA processed

by nucleases and by nucleases and small segments small segments then target RNase then target RNase to complementary to complementary RNA moleculesRNA molecules

Works well in plants Works well in plants and many animalsand many animals Not humansNot humans

Interfering RNAInterfering RNA The addition of dsRNA to an animal cell causes The addition of dsRNA to an animal cell causes

the degradation of the mRNA from which it is the degradation of the mRNA from which it is derived.derived.

This process is called gene silencing or RNA This process is called gene silencing or RNA inference (RNAi).inference (RNAi).

Gene silencing has been shown to be a natural Gene silencing has been shown to be a natural mechanism which plant and animals use to protect mechanism which plant and animals use to protect against viruses.against viruses.

The dsRNA that is introduced is cleaved by dicer-The dsRNA that is introduced is cleaved by dicer-like dsRNAse into ssRNA of 21-23 nt.like dsRNAse into ssRNA of 21-23 nt.

These short oligos complex with RISC ( RNA These short oligos complex with RISC ( RNA inference inducing silencing complex) which inference inducing silencing complex) which degrade the mRNA complimentary to the oligos.degrade the mRNA complimentary to the oligos.

This process can be used to target specific mRNA.This process can be used to target specific mRNA.

RNAi as Therapeutic AgentsRNAi as Therapeutic Agents

A viral vector was used to deliver a small A viral vector was used to deliver a small fragment of RNA to brain cells of mice with fragment of RNA to brain cells of mice with SCA1 (human neurodegenerative disease SCA1 (human neurodegenerative disease spinocerebellar ataxia 1).spinocerebellar ataxia 1).

This suppress the SCA1 gene and the mice This suppress the SCA1 gene and the mice has normal coordination and movement.has normal coordination and movement.

Scientists are optimistic about using RNAi Scientists are optimistic about using RNAi to treat other neurological diseases such as to treat other neurological diseases such as Alzheimer’s and Hunting’s disease.Alzheimer’s and Hunting’s disease.

3B. Gene Therapy3B. Gene Therapy

Strategies for Treating Genetic DisordersStrategies for Treating Genetic Disorders

Gene Therapy Clinical Trials Gene Therapy Clinical Trials 1990 - 19991990 - 1999

Production of Gene-encoding VectorsProduction of Gene-encoding Vectors

Prodrug ActivationProdrug Activation

Cells of tumor often Cells of tumor often interconnected by cytoplasmic interconnected by cytoplasmic bridges and poresbridges and pores

Introduce expression vector Introduce expression vector into some tumor cellsinto some tumor cells

Gene expressed converts Gene expressed converts prodrug into lethal compoundprodrug into lethal compound

““Shared” with other Shared” with other interconnected tumor cellsinterconnected tumor cells

““Bystander effect”Bystander effect”

Prodrug Activation Systems With a Prodrug Activation Systems With a Bystander EffectBystander Effect

THE END

HIV Therapeutic AgentsHIV Therapeutic Agents

Acquired immune deficiency syndrome Acquired immune deficiency syndrome (AIDS) is caused by the human (AIDS) is caused by the human immunodeficiency virus (HIV).immunodeficiency virus (HIV).

The target of HIV are the The target of HIV are the T helper cellsT helper cells (T (THH).). TTH H cells play a pivotal role in the immune cells play a pivotal role in the immune

system by the release of system by the release of cytokinescytokines which which stimulate other immune cells.stimulate other immune cells.

The The gp120 glycoproteingp120 glycoprotein of HIV binds to CD4 of HIV binds to CD4 receptors of Treceptors of THH cells. cells.

The TThe THH cells become infected with the virus cells become infected with the virus and are destroyed, slowly and are destroyed, slowly shutting down the shutting down the immune systemimmune system..

Interaction of HIV with CD4Interaction of HIV with CD4

HIV Therapeutic AgentsHIV Therapeutic Agents HIV antiviral strategies may include:HIV antiviral strategies may include: Production of Production of antibodies to CD4antibodies to CD4 (will block CD4 (will block CD4

receptors on Treceptors on TH H cells and prevent infection by HIV).cells and prevent infection by HIV). Production Production excess CD4 proteinexcess CD4 protein (react with gp120 (react with gp120

protein therefore HIV cannot infect Tprotein therefore HIV cannot infect THH cells). cells). Both strategies do not destroy HIV but only block Both strategies do not destroy HIV but only block

infection.infection. To stop HIV infection we need to develop strategies To stop HIV infection we need to develop strategies

which will destroy HIV.which will destroy HIV.

HIV Therapeutic AgentsHIV Therapeutic Agents

One strategy which will protect TOne strategy which will protect THH cells and cells and destroy HIV include the production of a destroy HIV include the production of a fusion fusion proteinprotein..

The fusion protein will have 2 parts The fusion protein will have 2 parts CD4 proteinCD4 protein attached to the Fc portion of an attached to the Fc portion of an immunoglobulinimmunoglobulin ( (CD4 immunoadhesionCD4 immunoadhesion).).

The CD4 portion will attach to the gp120 protein The CD4 portion will attach to the gp120 protein of HIV or virus infected cells.of HIV or virus infected cells.

The The immunoglobulinimmunoglobulin portion will initiate a portion will initiate a cytotoxiccytotoxic response to destroy the virus or virus response to destroy the virus or virus infected cell.infected cell.

CD4 Immuno-CD4 Immuno-adhesion Fusion adhesion Fusion

ProteinProtein

HIV Therapeutic AgentsHIV Therapeutic Agents

Another strategy involves making a second fusion protein.Another strategy involves making a second fusion protein. The The CD4 sequenceCD4 sequence is ligated to the sequence of is ligated to the sequence of

PseudomonasPseudomonas exotoxin A exotoxin A to form a fusion protein. to form a fusion protein. HIV infected cells have gp120 proteins on their surfaces.HIV infected cells have gp120 proteins on their surfaces. The CD4 portion of the fusion protein will attach to the The CD4 portion of the fusion protein will attach to the

infected cells.infected cells. The fusion protein will enter the cells and initiate the killing The fusion protein will enter the cells and initiate the killing

of the infected cell.of the infected cell. PseudomonasPseudomonas exotoxin A exotoxin A inactivates the protein inactivates the protein

synthesis by affecting elongation factor EF-2. This synthesis by affecting elongation factor EF-2. This prevents further protein synthesis and eventually causes prevents further protein synthesis and eventually causes death of the infected cell.death of the infected cell.

CD4-CD4-PseudomonasPseudomonas Exotoxin Fusion Exotoxin Fusion ProteinProtein