Protein Modifications

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Protein Modifications
Change in pharmacokinetics/pharmacodynamics May
alter protein stability/activity May cause
conformational changes in protein
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Types of Protein Engineering/Modifications

• PEGylation
• Amino acid substitutions
• Glycosylation
• Deletions and additions
• Fusion proteins
• Humanization (Mabs)

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Why PEGylate Proteins?
Protein drugs have relatively short
half-lives wide tissue distribution potential
for immunogenicity Means frequent dosing or
continuous infusion required increased
cost complicated dosing regimens
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What is PEGylation?
Chemical modification of proteins - addition
of monomethoxypolyethylene glycol polymer aka
PEG - PEG units added to enlarge the
protein - helps to prolong time in the body -
may lower immunogenicity
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PEGylated Protein Examples

• IL-2
• PEG IL-2 - has 1-5 PEG polymers attached
  via lysines
• Causes a decrease in elimination clearance
• rhIL-2 CL 4.07 ml/minkg
• PEG rhIL-2 CL 0.28 ml/minkg
• G-CSF
• Filgrastim (Neupogen) - modified by
  PEGylation
• ---gt Neulasta (new drug on market from Amgen)
• Interferon Alpha 2a
• Roferon modified by PEGylation
• ---gt PEGASYS (peginterferon alpha 2a)
• provides sustained serum levels for a full week

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Interferon alpha 2a (Roferon/PEGASUS)
Simple Branched
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(No Transcript)
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Protein Engineering
Alteration of the native protein sequence to
change or improve function Two basic
methods 1) Site-directed mutagenesis 2)
Fusion/hybrid/chimeric proteins
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Site-Directed Mutagenesis
To introduce changes at the DNA level that will
1) change amino acid codon -gt alters
protein 2) create new restriction enzyme
sites -gt facilitates manipulation of DNA
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Site-Directed Mutagenesis
1) To alter a single amino acid residue by
mutating the codon that encodes for that amino
acid. ATG GCC GGA GAC GAG ACT ACT AAA ATG GCC
GGA GTC GAG ACT ACT AAA translates
to.. Met - Ala - Gly - Asp - Glu - Thr - Thr
-Lys Met - Ala - Gly - Val - Glu - Thr - Thr
-Lys
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Site-Directed Mutagenesis
2) To create a new restriction site for
manipulation of DNA without introducing an amino
acid change. AAT TCG CAT TCT ATG GGT ACC Asn-
Ser - His - Ser - Met - Gly -Thr
NcoI AAT TCG CAT T(CC ATG G)GT
ACC Asn- Ser - His - Ser - Met - Gly -Thr New
restriction site, no change in amino acid
sequence. TCT Ser, TCC Ser
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Protein Engineering

• Change biochemical feature/activity of the
  protein
• Improve stability
• Add/remove glycosylation sites
• Aid in purification
• Understand how protein functions

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Altering Glycosylation Sites
Use site-directed mutagenesis to introduce new
glycosylation sites. Erythropoietin as an example
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In vivo Efficacy of EPO Isoforms
Br. J. Cancer (2001) 84 (Supplement 1), 3-10
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Aranesp - A new EPO
Erythropoietin direct relationship between
carbohydrate content (sialic acid) and its serum
half life and biological activity in
vivo Inverse relationship with receptor binding
(i.e., more glycosylation means poorer
binding) Hypothesis was the more
glycosylation --gt longer half life
--gt more activity --gt reduced
binding affinity
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Aranesp - A new EPO
Introduced new glycosylation sites to contain two
more N-linked sites than hEPO Hyperglycosylated
rhEPO biochemically distinct greater
molecular mass greater negative charge In
vivo no loss of drug function increased serum
half-life (3-fold longer) ---gt reduced
frequency of administration FDA approved 1/30/03
for anemia
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Fusion or Hybrid Protein
A novel protein engineered by fusing the protein
coding sequence of one gene to the protein coding
sequence of a different gene.
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Fusion Proteins
Can be used to direct therapeutic proteins or
genes to correct target. Can be used to direct
toxins to a target site.
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Fusion Proteins
Can be used to direct therapeutic proteins or
genes to correct target.
Mostly used in gene therapy to target
particular cells or tissues
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Fusion Protein
Can be used to direct toxins to a target site.
IL-2 diptheria toxin aa 2-133 human IL-2
1-389 aa of diptheria toxin Targets IL-2
receptors on CTCLs to kill them Denileukin
diftitox (Ontak) - FDA approved for cancer 30
patients have 50 reduction in tumor
burden CTCL cutaneous T-cell lymphoma
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Recombinant IL-2 continued
Ontak - IL-2 diptheria toxin fusion
protein Ontak binds to surface of lymphoma cells
via IL-2 receptor Once internalized, diptheria
toxin kills cell.
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Fusion protein created by fusing the second gene
downstream from the first gene
ATG
TAA
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3
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Fusion Protein Considerations
Removal of stop codon from first gene Coding
regions must be in-frame when fused Fusion
proteins are often not properly folded Can
introduce extra spacer amino acids
to facilitate proper folding
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Treatment of Rheumatoid Arthritis with a Tumor
Necrosis Factor Fusion Protein
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Normal Rheumatoid Arthritis
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Rheumatoid Arthritis
Autoimmune disease the immune system cannot
differentiate self from non-self tissue and
attacks the synovial and other tissues in the
joints. Inflammation and release of cytokines
in the joints. One major cytokine is tumor
necrosis factor or TNF.
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Tumor Necrosis Factor (TNF)
Cytokine released by activated macrophages and T
cells. Involved in the inflammatory response.
Excessive concentrations of TNF contributes to
the pathogenesis of autoimmune inflammatory
diseases.
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Macrophage
In RA excess TNF is generated This stimulates an
inflammatory response that results in cartilage
destruction
TNF
T Cell
Inflammation
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TNF interacts with the TNF receptor
TNF receptor exists asa plasma membrane receptor
Cytosol
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Want to make a fusion protein One portion
contains the ligand binding domain of the
membrane receptor
AUG
UAA
mRNA
5
3
N
Extracellular Receptor
Lipid Bilayer
Cytosol
C
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Soluble TNF receptor (sTNFR)
N

• Soluble portion of ligand binding domain present
  in body fluids.

C
Acts as a decoy, preventing TNF from binding to
its receptor.
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Goal
Use the sTNFR as a drug that will bind
TNF. Decrease serum TNF and reduce inflammation
in rheumatoid arthritis patients.
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Problem with recombinant sTNFR

• Not very stable in vivo.

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To circumvent these problems a TNF fusion protein
was created.
Soluble dimeric human TNF receptor Linked to the
Fc region of human IgG1
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sTNFRFc Fusion Protein
-S-S-
-S-S-
-S-S-
S
S
Soluble TNF Receptor
S
S
S
S
S
S
Fc region of IgG1
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Macrophage
TNF
TNF
TNF
T Cell
Inflammation
sTNFRFc
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Mechanism of Action
TNF

TNF
Excess TNF is bound to the soluble
receptor. This prevents TNF from binding to the
cells TNF receptors thereby reducing the
inflammatory response.
TNF
TNF
TNF
TNF
TNF
TNF receptors
cell
sTNFRFc
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Clinical trial of TNFRIgG1 Fusion Protein
(TNFRFc) N Eng J Med, 337141 (1997)
180 rheumatoid arthritis patients received twice
weekly injections of TNFRFc or placebo for three
months. After 3 months, symptoms of disease were
evaluated. 75 of patients in treatment group had
improvements in symptoms. 20 of the
placebo-treated group had improvements in
symptoms.
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ENBRELTM (etanercept)
Soluble Tumor Necrosis FactorReceptorIgG1
Fusion Protein
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Top 20 Biotech Products by Sales
Top 50 Biologicals in 2000 20 B Above 33
Products in 2002 26 B
Source Med Adv News, Vol.20 (5), 2001 most
drugs are 2002 sales noted by an , based on 2003
web sites Sales dollars are in millions
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Biological Response Modifiers

• Endogenous proteins stimulate the formation of
  blood cells during hematopoiesis
• Through rDNA, possible to produce quantities
  necessary to treat patients
• Reduce infections in patients with AIDS,
  infectious diseases and cancer chemotherapy

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Hematopoietic Cell Growth Factors
Soluble mediators that aid communication between
cells (primarily immunological, hematological,
neurological) Glycoproteins 60 different
cytokines Targeted to produce local
effects Brief, self-limiting effect
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Hematopoietc Cell Growth Factors
Erythropoietin Colony stimulating factors (G-CSF,
etc.) Interleukins Interferons Chemokines
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Pluripotent Stem Cell

• Most primitive blood-forming cell.
• All blood cells develop from this master cell.
• Located primarily in bone marrow (some in blood
  spleen).

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The Hematopoietic Cascade
Lymphoid
Myeloid
Granulocytes
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Life Span of Blood Cells

• Erythrocyte 120 days
• Platelet 7-10 days
• Monocyte 3 days
• Neutrophil 7-12 hours
• Lymphocyte 3 months to 10 years

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Role of Hematopoietic Growth Factors

• Regulates the proliferation and differentiation
  of progenitor cells.
• Proliferation (reproduction of similar cells)
• Differentiation (acquisition of function)
• Mobilize progenitor cells to move from bone
  marrow to peripheral blood.
• Regulates the survival and function of mature
  hematopoietic cells.

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Approved Biological Products Growth Factors
Generic Name Brand Name Company Therapeutic
Area (1st Date of Approval) Epoetin
alfa EPOGEN (Amgen) Certain anemias from
chronic renal disease (1989) Procrit
(Ortho Biotech) Zidovudine-induced anemia
(1991) cancer (1993) Anemia in
chemotherapy(1993) surgery patients
(1996) Filgrastim NEUPOGEN
(Amgen) Neutropenias due to myelosuppressive
(r-metHuG-CSF) chemotherapy (1991) Myeloid
reconstitution after bone marrow
transplantation (1994) Severe chronic
neutropenia (1994) Peripheral blood
progenitor cell transplant (1995) Induction
and consolidation therapy in AML (1998)
Sargramostim Leukine (Berlex) Myeloid
reconstitution after bone marrow
(GM-CSF) transplantation (1991) Bone marrow
transplant failure (1994) Adjunct to
chemotherapy in AML (1995) Peripheral blood
progenitor cell transplant (1995) Darbepoetin
alfa Aranesp (Amgen) Anemia associated with end
stage renal disease and (NESP) chronic renal
insufficiency (2001) cancer (2002) Becaplermin
(PDGF) Regranex (Ortho-McNeil) Diabetic foot
ulcer (1997) Pegfilgrastim Neulasta (Amgen) Neu
tropenias and infections due to
myelosuppressive chemotherapy (2002)
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Erythropoietin

• Endogenous glycoprotein, produced in kidneys.
• Promotes survival stimulates division/differentia
  tion RBC precursors in bone marrow.

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Epogen Procrit

• Recombinant human erythropoietin
• Glycosylated protein
• Same biological activity as endogenous
  erythropoietin
• Anemia associated with
• Chronic renal failure
• AZT-treated HIV patients
• Cancer patients on chemotherapy
• Surgeries (noncardiac, nonvascular) some blood
  loss, reduce need for blood transfusions.

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Aranesp (darbopoetin alpha)
Modified recombinant erythropoietin Engineered
for additional glycosylation sites Increased
serum half life
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GM-CSF and G-CSF Stimulate Hematopoietic Cells
GM-CSF and G-CSF

• Sargramostim (GM-CSF), Leukine (Berlex)
  Stimulates granulocytes (mostly
  neutrophils) monocytes
• Filgrastim (G-CSF), Neupogen (Amgen)
• Stimulates granulocytes (mostly neutrophils)
• Pegfilgrastim (G-CSF), NeulastaTM (Amgen)

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The role of GM-CSF versus G-CSF
GM-CSF
G-CSF
GM-CSF
GM-CSF
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GM-CSF (Sargramostim) Broad Acting Factor

• Complementary activity with G-CSF
• Involved in activating neutrophil production
• Enhances neutrophil chemotaxis and serum half
  life
• Stimulates macrophage production

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Sargramostim (GM-CSF) approved indications

• Chemotherapy induced neutropenia in patients with
  acute myelogenous leukemia (AML).
• Mobilization of stem cells following stem cell
  transplantation.
• Myeloid recovery following autologous bone
  marrow transplantation.
• Myeloid recovery allogeneic bone marrow
  transplantation.
• Bone marrow transplantation failure.

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G-CSF (Filgrastim)

• Involved in maintaining neutrophil production and
  in increasing production during infection or
  other stimulation.
• Targets granulocytes, mostly neutrophils.

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Filgrastim (G-CSF) approved indications

• Cancer patients receiving myelosuppressive
  chemotherapy.
• Cancer patients receiving bone marrow
  transplantation.
• Patients undergoing peripheral stem cell
  harvesting.
• Severe chronic neutropenia.
• Prevention of infection in AIDS patients.

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G-CSF/GM-CSF are used to treat infections

• Neutropenia secondary to chemo/radiation therapy
• Bone marrow transplantation
• Infectious disease

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Neutropenia from chemo/radiation therapy

• Neutrophils are the shortest-lived blood cells
• (7-12hrs).
• Most susceptible to toxicity from therapy.
• Both G- and GM-CSF stimulate neutrophil
  production.

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Advantages of G-CSF/GM-CSF in chemo-induced
neutropenia

• Reduce infections secondary to the toxic therapy
• Allow for dosage escalation of chemo/radiation
• kill more of the cancer

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Interleukins

• IL-1 ---gt IL-18
• Varied activities
• 2 commercial products
• Aldesleukin (IL-2) Proleukin (Chiron)
• Oprelvekin (IL-11), Neumega (Genetic Institute)

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Recombinant IL-2
IL-2 - made by activated T cells and induces T
cell proliferation and differentiation. Aldesleuk
in (Proleukin)- recombinant IL-2 produced in E.
coli, not glycosylated single aa change cysteine
125 -gt serine Approved in 1998 for treatment of
metastatic melanoma. Mechanism of action not
entirely clear - activation of T cells and
natural killer cells thought important.
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Recombinant IL-11
IL-11 is a thrombopoietic growth factor -
stimulates the proliferation of hematopoietic
stem cells and megakaryocyte proliferation and
maturation - leads to increased platelet
production Recombinant IL-11 - oprelvekin
(Neumega) produced in E. coli non-glycolylated
lacks N-terminal proline residue otherwise same
as native IL-11 no loss of biological
activity Used to reduce need for platelet
transfusions following myelosuppressive
chemotherapy
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Approved Biological ProductsInterferons
Generic Name Brand Name Company Therapeutic
Area (1st Date of Approval) Interferon
alfa-n1 Wellferon (GlaxoSK) Chronic hepatitis C
(1999) Interferon alfa-2a Roferon-A (Roche) Hai
ry cell leukemia (1986) AIDS-related Kaposi's
sarcoma (1988) CML (1997) Interferon
alfa-2b Intron-A (Schering) Hairy cell leukemia
(1986) AIDS-related Kaposi's
sarcoma (1988) Chronic hepatitis, types B
(1992) and C (1991) Condylomata acuminata
(1988) Malignant melanoma (1995) NHL
(1997) Interferon alfa-n3 Alferon-N (Interferon
Condylomata acuminata (1989) Sciences) Inter
feron gamma-1b Actimmune (InterMune) Chronic
granulomatous disease (1990) Osteoporosis
(2000) Interferon beta-1b Betaseron (Berlex) Acu
te relapsing-remitting multiple sclerosis
(1993) Interferon beta-1a Avonex (Biogen) Acute
relapsing-remitting multiple sclerosis
(1996) Rebif (Serono)
(2002) Interferon alfacon-1 Infergen (InterMu
ne) Hepatitis C (Naive and Relapse )
(1997) Peg-Interferon-2a Pegasys
(Roche) Hepatitis C (2000) Peg-Interferon-2b PEG
-Intron A (Schering) Hepatitis C (2001)