THERAPEUTIC AGENTS

1. Recombinant Proteins

2. Nucleic Acids

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 Therapeutic Agents
Introduction

Process of Drug Production

Formulation/
Transfection Cell culture Purification
Filling

Drug
Cells and plasmid Cell line Drug Drug product -
substance substance (sterile)
(crude) (pure)

Cell line manufacture Bioreactor process Downstream

Medium development development & scale-up purification

Analytical characterization 2
 Therapeutic Agents
Introduction
Development of the Process

Biological Assay Development and Support

Cell Line/ Cell/Virus Purification Formulation

Viral Vector/ Culture Process Design/
Construction Development/ Dev. Drug Delivery
Media Design
Optimization

Facility/Equipment Design
Technology Transfer
Process Validation
Pilot-Scale cGMP Production
Commercial-Scale Production
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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections
-> Scientists discovered an antiviral protein in 1957 that inhibited
growth of influenza virus in chicken embryos. It was named interferon
because it interfered with the growth of influenza virus.

• Anti viral proteins released by host cells (part of the immune system)

• Interfere with viral multiplication

• Host cell specific but not virus specific

• Different types of cells in animals produce different interferons

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections

• 3 types of human interferon:

– alpha interferon (13 genes)
– beta interferon (2 genes)
– gamma interferon (1 gene)

• Alpha & beta usually produced early in viral infections (viruses or viral
RNA) - Gamma appears later

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

-> Viral infected cells release alpha and beta interferons

Diffuse to neighboring cells -> Virus can’t replicate

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections

Antiviral Treatment:

• Interferon therapy
– Limited lifetime, short lasting effect

– Recombinant interferons
• Pure and fast
• Hybrid genes for enhanced/new activity

– Oral administration

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections

Why are Side Effects Common and Severe for Injectable Interferon?
• Injectable interferon (beta) is approved world-wide (FDA) for the
treatment of various cancers and viral diseases.
• Interferon is a protein readily eliminated from the blood by the kidney. To
counteract the kidney’s clearance of interferon from the blood injectable
interferon must be given in doses much higher than what occur naturally.
• Side effects include flu-like symptoms, poor results on liver function tests,
and blood cell abnormalities. More serious side effects include depression,
epileptic seizures, or liver problems.

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 Why is Oral Interferon Different?

• Low-dose oral interferon is given in doses 10 thousand times less than

injectable interferon. Therefore, side effects are dramatically
reduced.
• Oral interferon is human interferon alpha administered in a small
tablet (lozenge) to humans or in powder to animals.
• Oral interferon binds to surface (mucosal) cells in the mouth and
throat resulting in stimulation of white blood cells and activates
hundreds of genes affecting the immune system in the peripheral blood
of man, cattle and mice.
• Studies show oral interferon is effective against disorders such as
cancer, viral diseases and autoimmunity.

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections

Mechanism of Action Oral

Epithelial
Cells
Tonsil
IFNa

Mandibular Lymph Nodes

Activation of Perioral Lymphoid Cells and

Activation of Peripheral Lymphoid Tissues
Humoral
Immunity
(Antibody) Activation of
Virus Cell Mediated
Immunity

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.

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Manufacturing Steps for Interferon:

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections

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

Behcet's disease Chronic active hepatitis B

Sjogren's syndrome
Idiopathic pulmonary fibrosis
Multiple sclerosis
Aphthous stomatitis
Oral mucositis - cancer
Lichen planus
Opportunistic infections - HIV+
Chronic hepatitis C
Wasting cancer patients
Fibromyalgia
Measles
Respiratory syncytial virus
Influenza
Cough (COPD & IPF)

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 Therapeutic Agents
Recombinant Proteins
Human Interferons -> to fight viral infections
Human Study – Influenza and Interferon

• 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.

Efficacy of Human Leukocyte Interferon as

Prophylaxis Against Influenza
25
Percent of Patients Sick

20

15
Interferon
10

5 Placebo

0
Adults Children Children
7-12 yr 2-6 yr Soloviev, Bull. WHO 41:683-688, 1969. 12
Population (14,000 subjects total)
 Therapeutic Agents
Recombinant Proteins

Strategies for Optimisation of Recombinant Production

• Screening libraries of recombinant genes (IFNs, human growth

hormone, TNF-a…)

• Screening of recombinant expression systems (E coli, fungi,

Mammalian cells…)

• Delivery by intestinal bacteria (lactobacilli)

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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

• Cystic fibrosis (CF) -> ~30,000 cases in the US and 23,000 in Canada.
• Europe: it occurs in 1 in 2,500 live birth and 1 in 25 are carriers.
• Caused by more than 500 different mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene.
• Individuals with CF are highly susceptible to bacterial infection and antibiotic
treatment often results in resistant strains.

Symptoms: -> small ducts (special channels) become clogged with

a thick mucus
• Clogging and infection of lungs
• plugging of small bile ducts in liver (impedes digestion)
• plugging of ducts of pancreas (impedes digestion)
• obstruction of small intestine
• males are infertile
• malfunctioning sweat glands
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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

Gene responsible for disease:

CF is mostly caused by a 3 base

pair deletion in Cystic Fibrosis
Transmembrane Regulator
(CTFR)
-> F508 deleted
CTFR -> ABC transporter
coupled to a channel

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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

-> It transports Cl- ions after being

phosphorylated and binding two ATP
molecules

-> It has a large regulatory domain that

is phosphorylated by a cAMP dependent
protein kinase

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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

A normal lung
Chloride into airway;
sodium out - keeps
mucus moist and thin
Normal CFTR
regulates the sodium
channel (inactivates it)

A CF lung
Chloride does not get
into airway; more
sodium leaves; More
salt in cell -> water
comes in ->This makes
the mucus thick

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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

Treatments:

• Chloride delivery - activate other chloride carriers

• Viscous mucus - pounding, DNase treatment, gelosin

• recurrent infections – antibiotics

• tissue damage due to immune response - anti-inflammatory drugs

(ibuprofen)

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 Therapeutic Agents
Recombinant Proteins

Enzymes – Treating Cystic Fibrosis

DNase 1 (GeneTech)
• A thick mucus which is a results of:
– Alignate produced by bacteria
– DNA from lysed cells
– Leucocytes which accumulate due to the infection
• Makes breathing difficult.
• Scientist at Genentech isolated the gene for DNase1
• The purified enzyme was delivered as an aerosol to the lung where it hydrolysed the DNA into
short oligonucleotides.
• This decrease the viscosity in the lungs and made breathing easier.

Alginate Lyase
• Alginate is a polysaccharide polymer that is produced by bacteria.
• The excretion of alginate by Pseudomonas aeruginosa of patients with CF contributes to the
viscosity in the lung.
• The enzyme alginate lyase can liquefy bacteria alginate.
• Alginate lyase was isolate from Flavobacterium sp. and cloned into E. coli.

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

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 Therapeutic Agents
Recombinant Proteins

Monoclonal Antibodies
Clinical Applications
• Transplantation – muronomab (OKT3) 1986, basiliximab 1998

• Cardiovascular disease – abciximab 1994

• Cancer – rituximab 1997, trastuzumab 1998

• Viral infection – palivizumab 1998

• Inflammatory diseases – infliximab 1998, etanercept 1999

Side effects: • Transfusion reactions (any adverse event which occurs because of a blood
transfusion)
• Infections, immunosuppression
• Cardiac, respiratory arrest (discontinuation of breathing)
• Pharmacological toxicity
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 Therapeutic Agents
Recombinant Proteins

Monoclonal Antibodies
Production

• Monoclonal antibodies results from a clone of a B lymphocyte producing a

single antibody which will bind to a specific epitope of an antigen.
• Monoclonal antibodies are produced:
– Fusion of a myeloma (B cell which has become cancerous) with a
spleen cell that is immunized with a specific antigen.
• The resulting hybridomas are tested for the production of a monoclonal
antibodies.

• Diluted to one cell cultures

• Hypoxanthine Guanine Phosphoribosyl Transferase (HGPT) negative
myeloma cells
• Grown in Hypoxanthine Aminopterin Thymidine (HAT) medium
• Only successful fusion cells grow (rare)

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 Therapeutic Agents
Recombinant Proteins
Production: Monoclonal Antibodies

Major Problems with non-human ABs:

Immunological Responses

 ”Humanization” of ABs

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Chimeric Ab
65 – 90% human and consist
of the mouse variable regions
and substituting the mouse
Fc region of the antibody
with that from human
Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Humanized Ab
DNA that encodes the binding
portion of monoclonal mouse
antibodies and merges it with
human antibody-producing DNA.
Use mammalian cell cultures to
express this DNA and produce
these half-mouse and half-human
antibodies. (Bacteria cannot be
used for this purpose, since they
cannot produce this kind of
glycoprotein.) Depending on how big
a part of the mouse antibody is
95% human, and are made by used, one talks about chimeric
grafting the hypervariable antibodies or humanized
region (or CDR) of the antibodies.
chimeric antibody –which
determines Ab specificity
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 Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:
-> uses various "display" methods
(primarily phage display) as well as
methods that exploit the elevated B-
cell levels that occur during a human
immune response.

• Create new variations of antibodies

– New combinations of heavy and light
chains
• mRNA from immunized individual
• PCR H and L chains
• Clone into vector in new H/L
combinations

-> create library -> screening by display

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 Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:
Screening by Phage display

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 Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:

Single-chain Fixed variable

only one heavy-chain variable

domain and one light-chain variable
domain covalently linked by peptide
Single-Chain Combinatorial Antibody Library

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 Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Application of single-chain fixed variable-> Immunotoxins

• Protein toxin connected to Fv region

• Single-chain or S-S linked Fv region
• Toxin localized to antigen-expressing cells

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 Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Examples:

Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC,
antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb,
monoclonal antibody; scFv, single-chain Fv fragment 28
 Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Examples:
Target Angiogenesis -> to prevent tumor growth

Dr. Judah Folkman (1971)

Tumor secretes factors -> promote blood vessel growth to

the tumor
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 Therapeutic Agents
Recombinant Proteins
Examples: Monoclonal Antibodies
Bevacizumab (Avastin®)
Target Angiogenesis -> to prevent tumor growth

• Recombinant humanised monoclonal antibody target

ing the angiogenic factor VEGF (93% human, 7%
mouse)

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 Therapeutic Agents
Nucleic Acids

As Therapeutic agents

• Many human disorders e.g. cancer and inflammatory conditions (virus,

parasites) are often caused by overproduction of a normal protein.

• Theoretically a small ss nucleic acid can hybridize to a specific gene or

mRNA and diminish transcription or translation. (antisense RNA)
• An oligonucleotide (oligo) that binds to a gene and blocks transcription is
an antigene.
• An oligo that binds to mRNA and blocks translation is called an antisense
oligo or antisense RNA.
• Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific
mRNA for degradation.

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 Therapeutic Agents
Nucleic Acids

Antisense RNAs

• Gene segment cloned into

expression vector in
reverse orientation

• Formation of dsRNA can

interfere with RNA
processing or translation

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 Therapeutic Agents
Nucleic Acids

Antisense RNAs -> Applications

• Episomally based expression vectors with cDNA for insulin-like growth factor 1
(ILGF-1) receptors were constructed in the antisense version.
• ILGF-1 is prevalent in malignant glioma a common form of brain cancer and
prostate carcinoma.
• Culture of glioma cells when transfected with the antisense version of ILGF-1 in
ZnSO4 lost its tumurous properties.
• A similar treatment of mice which were injected with prostate carcinoma cells
caused small or no tumor to develop.

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 Therapeutic Agents
Nucleic Acids

Antisense Oligonucleotides
• Antisense deoxynucleotides can also be used as therapeutic agents.
• However when injected into the body is deoxynucleotides are susceptible to
degradation.
• To prevent this modified deoxynucleotides are used including phosphorothioate,
phosphoramidate and polyamide.
• Free oligos are usually introduced into to the body encapsulated in a liposome.

Phosphodiester Phosphorothioate
linkage linkage Phosphoramidite Polyamide
linkage linkage
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 Therapeutic Agents
Nucleic Acids

Antisense Oligonucleotides
• Antisense deoxynucleotides can also be used as therapeutic agents.
• However when injected into the body is deoxynucleotides are susceptible to
degradation.
• To prevent this modified deoxynucleotides are used including phosphorothioate,
phosphoramidate and polyamide.
• Free oligos are usually introduced into to the body encapsulated in a liposome.

Liposome Delivery of Therapeutic Nucleic Acids:

• Small vesicles of phospholipid bilayer
• Fuse to cells and release contents into cytoplasm
• Approach can target human cells and/or infectious
agent in early stage tests (e.g. mycobacterium
tuberculosis)

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 Therapeutic Agents
Nucleic Acids

Antisense Oligonucleotides
Treatment of Psoriasis:

• Psoriasis is uncontrollable epidermal growth.

• ILGF-1 receptors are implicated (upregulated) in the pathogenesis of psoriasis.
• 15 nt antisense oligo were transferred into keratinocytes using liposome and the amount of
ILGF-1 protein was decreased by 45-65%.
• When mouse with human psoriasis lesions were injected with anitsense oligi complementary to
ILGF-1 receptor mRNA there was significant reduction (58-69%) in epidermal thickness.

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

• Synthetic oligonucleotide blocks site of mutation which

created mutant “alternative” splice junction
– Acts at RNA level

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 Therapeutic Agents
Nucleic Acids

RNA Interference (RNAi)

• RNA interference or gene silencing
• dsRNA processed by nucleases into small
segments then target RNase to
complementary RNA molecules
• Works well in plants and many animals
-> Not humans

• Gene silencing has been shown to be a natural

mechanism which plant and animals use to
protect against viruses.
• The dsRNA that is introduced is cleaved by
dsRNAse into ssRNA of 21-23 nt.
• These short oligos complex with RISC ( RNA
inference inducing silencing complex) which
degrade the mRNA complimentary to the
oligos.
• This process can be used to target specific
mRNA.

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 Therapeutic Agents
Nucleic Acids

RNA Interference (RNAi)

As Therapeutic Agents:

• A viral vector used to deliver a small

fragment of RNA to brain cells of mice
with SCA1 (human neurodegenerative
disease spinocerebellar ataxia 1).
• This suppress the SCA1 gene and the
mice has normal coordination and
movement.
• Scientists are optimistic about using
RNAi to treat other neurological diseases
such as Alzheimer’s and Hunting’s
disease.

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 Therapeutic Agents
Nucleic Acid

Gene Therapy – Clinical Trials (1990-1999)

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 Therapeutic Agents
Nucleic Acid

Gene Therapy – Principle

• Cells of tumor often

interconnected by
cytoplasmic bridges and
pores
• Introduce expression
vector into some tumor
cells
• Gene expressed converts
prodrug into lethal
compound
• “Shared” with other
interconnected tumor
cells

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 Therapeutic Agents
Nucleic Acid

Gene Therapy – Prodrug Activation Systems

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