Immunocamouflage (PEGylation)
of Red Blood Cells
Mark D. Scott, Ph.D.
Senior Scientist & Clinical Professor
Associate Director, Intellectual Property and Business Development
The Canadian Blood Services
University of British Columbia
Center for Blood Research & Department of Pathology & Laboratory Medicine
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Mark D. Scott 04/2009
1
it’s in you to give
Canadian Blood Service
Red Blood Cells
 Platelets
 White Blood Cells
 Collection
 Distribution
Supply
RBC PROBLEMS: Lack of O- Blood,
Alloimmunization, Shelf-Life, Cost of
Production, Cost of Distribution
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The Holy Grail
Solving the Problems of Traditional Blood Products
Blood
Substitutes!
Don’t need (as many) donors!
No Blood Groups - carefree…!
Long shelf life - months...!
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it’s in you to give
“Blood Substitutes”
X
1980s-1990s
• Perflurocarbons
What Does The
Holy Grail
Look Like?
• Complement Activation
• Vascular Leakage
• Other RBC Functions?
X
1980s-2000s
• Purified
Hemoglobins
Free & Liposome Encapsulated
• Toxicity
• Small Size
• Vascular Leakage
• Tissue Iron Loading
• Other RBC Functions?
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A Brief History of Hb-Based
Blood Substitutes
1980 - 2008
1980s:
US Military BEGINS massive (and expensive) research
program to develop hemoglobin (Hb) based blood substitutes.
Numerous private companies (e.g., Somatogen, Baxter, Hemosol,
Northfield, BioPure) begin commercialization of yeast-produced,
human and bovine based Hb-based blood substitutes.
1990s:
US Military STOPS massive (and expensive) research
program to develop Hb-based blood substitutes. Private companies
“soldier on” trying to produce a safe and commercially viable product.
Many fail or abandon development (e.g., Somatogen and Baxter).
2000s:
Despite two decades of commercial (e.g., Hemosol ) and
clinical trial failures, a few companies persist in development of Hbbased blood substitutes (Sangart, Northfield and BioPure).
Commercial success and product safety remain uncertain.
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BioPure (BPUR)
Produces BOVINE sourced Hemopure (human
use) and Oxyglobin (veterinary use).
Hemopure: approved for sale in South Africa for surgical patients, anemic patients,
and for eliminating, delaying, or reducing allogenic red blood cell transfusions in
these patients. [Problems with repeated dosing....]
Oxyglobin: oxygen therapeutic approved by the US FDA and the European
Commission for the treatment of anemia in dogs. The Company has sold ~182,000
units of Oxyglobin since approval.
030303
$288.00
Cost to transfuse a
“Yellow” Lab once*:
~$1000.00
*At the recommended dosage
090224
$0.08
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So Why Does Purified Hemoglobin Fail?
The job of blood is to deliver Oxygen to the tissues...
purified hemoglobin does this badly!
Vasoconstriction is
just ONE of the
multitude of problems
associated with
purified hemoglobin
solutions!
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In General, “Blood Substitutes” Have Been A
Major Disappointment
Perhaps Darwin Has A Point!
“Hemoglobin shall be INSIDE
red blood cells…” “except, of course,
in earthworms.”
The Book of Darwin:
Chapter 14, Verse 8
However, Darwin’s teachings requires
that the inherent antigenicity and
immunogenicity of the RBC be tamed.
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it’s in you to give
“Blood - It’s In You To Give®”
®Canadian
Blood Services
Perflurocarbons
Purified
Hemoglobins
Free & Liposome Encapsulated
Meeting Darwin’s Requirements....
Immunocamoulfaged
RBC
Taming the inherent antigenicity and
immunogenicity of the RBC.
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Immunocamouflage of Cells
Since the mid-1970’s methoxypoly(ethylene glycol) [mPEG] has been
“glued” to proteins to prolong circulation and prevent immune
recognition. PEGylated enzymes are currently used for the clinical
treatment of enzymopathies.
My Laboratory Pioneered The
Immunocamouflage of Intact Cells
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Poly(Ethylene Glycol)
Carbide and Carbin Chemicals
Corporation (Union Carbide)
HO
( )
H
H
C
C
H
H
O
n
H
H
C
C
H
H
Carbowax®
In 1927 Prestone introduced Ethylene
Glycol* and the era of modern automotive
antifreeze begun (thus saving modern Canada).
*replacing such things as molasses, honey and methyl alcohol.
Poly(ethylene glycol) is repeating units of
automobile antifreeze!
?! That can’t be safe!?
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Mark
MarkD.
D.Scott
Scott 02/2009
04/2009
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it’s
it’s in
in you
you to
to give
give
OH
Is PEG Safe?
By chemically linking several ethylene glycol monomers
together, a non-toxic polymer [i.e., Poly(ethylene glycol)] is
produced that is commonly used in modern medicine.
Safe
Oral Rat LD50 for:
Monomer
4.7 gm/kg
PEG 200:
28 gm/kg
PEG 1450: 40 gm/kg
PEG 4000: >50 gm/kg
Deadly
~ 4 kg for me...
Safe
At very HIGH levels, PEG oral
toxicity arises due to GI “flow” effects…
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PEG: Safe and Non-Toxic
Shaffer, C., Critchfield, F. The absorption and excretion of the solid polyethylene glycols
("Carbowax" compounds). Journal of the American Pharmaceutical Association
36:152-157; 1947.
Smyth, H.; Carpenter, C., Shaffer, C. The toxicity of high molecular weight polyethylene
glycols; chronic oral and parental administration.
Journal of the American
Pharmaceutical Association 36:157-160; 1947.
1947 ORAL (10 g bolus):
Safe. No measurable absorption from
gut, but at high concentrations this
industrial “lubricant” did cause diarrhea.
INJECTION (1 g in 20 ml):
Safe.
“Gim’me da carbo or I’ll wax ya.”
~90% of PEG excreted by
kidneys within 12 hours.
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PEG
Industrial-Medical-Consumer Applications
Adhesives
Agriculture
Ceramics
Cosmetics and Personal Care
Electronics
Electroplating
Electropolishing
Food
Food Processing
Household Products
Lubricants
Metalworking
Paints and Coatings
Paper and Paper Products
Pharmaceuticals
Printing and Inks
Rubber
Elastomers
Textiles
Wood Treating
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How Does PEG Work
HO
( )
H
H
C
C
H
H
O
n
H
H
C
C
H
H
OH
PEG is a water-soluble, nontoxic, low immunogenicity polymer
readily cleared from the body. Approved for oral, intravenous,
intramuscular, subcutaneous and topical administration.
Physically
creating a
semi-dense
cloud above
the cell
surface
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it’s in you to give
How Does PEG Work
HO
( )
H
H
C
C
H
H
O
n
H
H
C
C
H
H
OH
PEG is a water-soluble, nontoxic, low immunogenicity polymer
readily cleared from the body. Approved for oral, intravenous,
intramuscular, subcutaneous and topical administration.
Physically
creating a
semi-dense
cloud above
the cell
surface
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Immunocamouflage of Cells
Hiding the “Weapons of Mass Destruction”
in Transfusion, Transplantation, and Family Medicine
BLOOD
TISSUES
SNOT
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RBC Structure,
Function, and In Vivo
Survival?
Do mPEG-RBC “Work”....
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Stealth Erythrocyte Are NORMAL...
or at least appear to be normal...
Normal Human RBC Parameters
PEGylated Murine RBC Exhibit Normal In
Vivo Survival
Lysis
Grafting Process Does NOT
Harm The RBC (<1% Lysis)
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Mark
25
Mark D.
D. Scott
Scott 04/2009
03/2009
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it’s in
to give
it’syou
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to give
Biophysical Effects of
mPEG Grafting
How Does the grafted mPEG
“Work”....
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STEALTH CELLS - Charge Camouflage
Cell Charge
Plays An
Important Role
In Immune
Recognition.
Modified from: Scott,
M.D. et al. (2000)
Transfusion Medicine
Reviews, 14:53-63.
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Cell-Cell Interaction: Erythrocyte Sedimentation
Erythrocyte Sedimentation Rate (mm)
35
Rouleaux Formation
BTC-mPEG 20 kDa
Modified From: Bradley,
A.J. et al. (2002)
Biochimica et Biophysica
Acta, 1561:147-58.
30
Control
0 mM
Untreated
25
20
RBC Specific Gravity = 1.095
Plasma Specific Gravity = 1.031
15
0.6 mM
1.2 mM
2 mM
3 mM
4 mM
5 mM
10
5
Charge Mediated
0
0
1
2
3
Hours
4
5
mPEG-RBC
6
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Cell-Protein Interaction: Decreased Fluorescent
Protein Adsorption to Bare or mPEG-Modified
Latex Particles (8 µm).
20 kDa
SVA-mPEG
Bare Latex
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Decreased Low Shear Viscosity: Utilization For Vascular
Occlusive Diseases (e.g., SCD)?
Like Syrup
Bad
Like Water
Good
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Immunocamouflage of the
Rh Blood Group Antigens
20 nm
10 nm
10 nm
The Rh antigen family
is located at the
membrane surface
does NOT extend very
far out.
RhD – making the
“Near” Universal
RBC
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Reducing The Risk Of Alloimmunization (Rh Family)
Single Exposure Risk of Alloimmunization
BLOOD
GROUP
ANTIGEN
Percent Immuno-
Hemolytic
genicity¢
Trans. Reaction
ABO
A,B
100ª
Yes
IgM
Rh
D
50.00
Yes
IgG
c
2.05
Yes
IgG
E
1.69
Yes
IgG
e
0.56
Yes
IgG
C
0.11
Yes
IgG
Jka
0.07
Yes
IgG
Jkb
0.03
Yes
IgG
Duffy
Fya
0.23
Yes
IgG
Kell
k
1.50
Yes
IgG
K
5.00
Yes
IgG
S
0.04
Yes
IgG
s
0.03
Yes
IgG
Kidd
MNS
Primary Ab
Polymer Grafting Effectively
Attenuates Antibody
2% Risk
Recognition of Rhc
Mean Cell Florescence
Derived from “Clinical Experimentation”
??% Risk
¢ Chance of AlloimmunizationFollowing Each Antigen
Positive Transfusion In A Null Individual
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Immunocamouflaged of RhD
Control
0.6 mM
1.2 mM
Agglutination
Test for
2.4 mM
RhD
5 mM
O+, O-, A+, A-, B+, B-, AB+, AB-
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Is PEGylated RhD Immunogenic?
RhD Allorecognition Using RhD- Antigen Presenting Cell (APC;
Dendritic-Like Cells) Cultures.
RhD-
Blood
Flow
Cytometer
Analysis
CFSE-Labeled Fresh
Autologous RhD- PBMC
Plasma
PBMC
Ficoll-Hypaque
RBC
Attach PBMC
(5hrs)
Mature
DC-Like
Immature
DC-Like
5
Days
2
Days
IL-4
GM-CSF
IL-4
GM-CSF
IL-1
IL-6
TNF-
PGE-2
RhD
Peptide
1 Hour
CD80
CD83
CD86
HLA-DR
(remove)
2-4
Days
CFSE
Proliferation
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Is PEGylated RhD Immunogenic?
RhD Allorecognition: RhD- PBMC Proliferation Following
RhD Peptide Presentation By RhD- APC at 48 Hours.
A
Proliferation
1.3%
No RhD
(Neg. Control)
B
Proliferation
C
Proliferation
0.5%
75%
RhD Peptide
(Pos. Control)
SVA-mPEG
RhD Peptide
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Immunocamouflage of RhD
Improving blood utilization and availability.
Reducing The “Flavors” of Blood?
Immunocamouflaged
8 Crucial Flavors RBC
Type O ~ 44%
Type A ~ 42%
Some Realatively Rare...
Type B
~ 10%
Type AB ~ 4%
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Stealth RBC: The Challenges
To Being Famous and Rich
$$
Corporate
Considerations
It is NOT the next Viagra
Drug Approval
Process
$$
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Immunocamouflage of Blood
What About Other Blood Cells?
BLOOD
Transfusion-Associated Graft Versus Host Disease
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Paris Hilton® School of Medicine:
IMMUNOLOGY 101
Multiple determinants on cells govern whether individuals may be
potential tissues donors or tissue recipients.
Antigens
Paris and Not Paris
Stop
PARIS
US
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Immunocamouflage:
Essen tia l Cos tim u la to ry
P a thw ay
T Cell
CD80
CD28
40000
(B 7 )
CD28+
1000
Control
FSC-Height
Unmodified
30000
27.12%
72.88%
0
20000
0
10 10
1000
FSC-Height
3H-Thymidine
Incorporation (CPM)
Loss of Allorecognition
APC
10000
1 .0
3
1 .5
2 .0
Responder Cell Concentration (x
2.4 mM mPEG
7
per 1 x 10 cells
99.80%
2.5
105)
100 101
102 103 10
C D 2 8-FI T C
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4
10
0
0
0 .5
2
10
10
C D 28-FI T C
0.20%
mPEG-Modified
0
1
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4
Global Immunocamouflage of Either the
APC or Lymphocyte Prevents Allorecognition
Both In Vitro and In Vivo
CONTROL
mPEG-MODIFIED
X
Allorecognition
X
Costimulation
X
Adhesion
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A Need To Innovate:
Immunocamouflage of Blood Cells is
Easily and Effectively Accomplished.
This is not a broken Menorah
Potential Applications
Include:
Reduce Risk of RBC Alloimmunization
Improved RBC Rheological Dynamics (Sickle Cell Anemia)
Improved Utilization of RBC (e.g., just say “NO!”toRhD)
Prevention of Transfusion Associated-GVHD
Cold Storage of Platelets
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Immunocamouflage Acknowledgments
STUDENTS &POSTDOCS
Amanda Bradley, Ph.D.
Audrey Chen, Ph.D.
YevgeniyaLe, Ph.D.
Kari Murad, Ph.D.
Troy Sutton, Ph.D.
Dan Wang, Ph.D.
TECHNICIANS
Nobu Nakane, M.S.
Janet Tong
Wendy Toyofuku
COLLABORATORS
John W. Eaton, Ph.D.
Elisabeth Maurer, Ph.D.
Marshal Henri Pétain
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it’s in you to give