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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Site-Directed Mutagenesis
Oligonucleotide with mutation (change) 1) Used
with PCR or 2) with ssDNA template to make
dsDNA plasmid
Primer 2
Template DNA
X
Primer 1
PCR
X
Mutated DNA
X
Clone into plasmid
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Site-Directed Mutagenesis
Oligonucleotide with mutation (change) 1) Used
with PCR or 2) with ssDNA template to make
dsDNA plasmid
Mutagenic oligo
x
x
x
dNTPS polymerase
ssDNA
dsDNA
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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
5
3
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Construction of a Fusion Protein (Gene)
Site-directed mutagenesis to introduce
restriction sites where needed Use molecular
cloning technology to cut and paste genes
together Clone into appropriate expression
vector Express fusion protein
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Creating a Fusion Gene
EcoRI
BamHI
TAA (stop)
ATG
ATG
TAA (stop)
Site-directed mutagenesis to introduce new sites
EcoRI
BamHI
HinDIII
HinDIII
TAA (stop)
ATG
ATG
TAA (stop)
Cut with HinDIII and fuse genes together
EcoRI
BamHI
HinDIII
ATG
TAA (stop)
Digest with EcoRI and BamHI Clone into plasmid
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ATG
TAA
5
3
C
Fusion Protein
N
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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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Amevive - Fusion Protein
For the treatment of psoriasis Psoriasis - a T
cell mediated autoimmune disease Activated T
cells secrete cytokines and incite an
inflammatory response Hypothesis If one could
prevent activation of the T cells, may be able
to reduce inflammatory response
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Cell Interactions Involved in Psoriasis
Activation of T lymphocytes through interaction
between LFA-3 on APCs and CD2 on T cells plays a
role in the pathophysiology of chronic psoriasis.
Blocking this interaction may reduce psoriasis.
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Amevive (alefacept) - Fusion Protein
Amevive is a fusion protein consisting of
extracellular CD2 binding portion of LFA-3
(leukocyte function antigen-3) linked to the Fc
portion of IgG antibody. Produced in CHO
cells MOA Amevive binds to CD2 and prevents the
interaction between LFA-3 and CD2
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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