Immunocamouflage (PEGylation) of Red Blood Cells

1
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
2
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
3
The Holy Grail
Solving the Problems of Traditional Blood Products
Dont need (as many) donors! No Blood Groups -
carefree! Long shelf life - months...!
4
Blood Substitutes
What Does The Holy Grail Look Like?
5
A Brief History of Hb-Based Blood
Substitutes1980 - 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 Hb-based
blood substitutes (Sangart, Northfield and
BioPure). Commercial success and product safety
remain uncertain.
6
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.
7
So Why Does Purified Hemoglobin Fail?
The job of blood is to deliver Oxygen to the
tissues... purified hemoglobin does this badly!
8
In General, Blood Substitutes Have Been A Major
Disappointment
9
Blood - Its In You To Give
Canadian Blood Services
10
Since the mid-1970s 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.
Immunocamouflage of Cells
11
Carbowax
Poly(Ethylene Glycol)
Carbide and Carbin Chemicals Corporation (Union
Carbide)
12
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.
At very HIGH levels, PEG oral toxicity arises due
to GI flow effects
13
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 36152-157
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 36157-160 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. 90 of PEG
excreted by kidneys within 12 hours.
Gimme da carbo or Ill wax ya.
14
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
15
How Does PEG Work
PEG is a water-soluble, nontoxic, low
immunogenicity polymer readily cleared from the
body. Approved for oral, intravenous,
intramuscular, subcutaneous and topical
administration.
16
How Does PEG Work
PEG is a water-soluble, nontoxic, low
immunogenicity polymer readily cleared from the
body. Approved for oral, intravenous,
intramuscular, subcutaneous and topical
administration.
17
Immunocamouflage of Cells
Hiding the Weapons of Mass Destruction in
Transfusion, Transplantation, and Family Medicine
18
RBC Structure, Function, and In Vivo Survival?
Do mPEG-RBC Work....
19
Stealth Erythrocyte Are NORMAL...
or at least appear to be normal...
25
20
Biophysical Effects of mPEG Grafting
How Does the grafted mPEG Work....
21
STEALTH CELLS - Charge Camouflage
Cell Charge Plays An Important Role In Immune
Recognition.
Modified from Scott, M.D. et al. (2000)
Transfusion Medicine Reviews, 1453-63.
22
Cell-Cell Interaction Erythrocyte Sedimentation
Modified From Bradley, A.J. et al. (2002)
Biochimica et Biophysica Acta, 1561147-58.
RBC Specific Gravity 1.095 Plasma Specific
Gravity 1.031
23
Cell-Protein Interaction Decreased Fluorescent
Protein Adsorption to Bare or mPEG-Modified Latex
Particles (8 µm).
20 kDa SVA-mPEG
24
Decreased Low Shear Viscosity Utilization For
Vascular Occlusive Diseases (e.g., SCD)?
.
25
Immunocamouflage of the Rh Blood Group Antigens
The Rh antigen family is located at the membrane
surface does NOT extend very far out. RhD
making the Near Universal RBC
20 nm
10 nm
10 nm
26
Reducing The Risk Of Alloimmunization (Rh Family)
Single Exposure Risk of Alloimmunization Derived
from Clinical Experimentation
BLOOD GROUP ANTIGEN Percent Immuno-genicity Hemolytic Trans. Reaction Primary Ab
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
Kidd 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
MNS S 0.04 Yes IgG
s 0.03 Yes IgG
Chance of AlloimmunizationFollowing Each
Antigen Positive Transfusion In A Null Individual
27
Immunocamouflaged of RhD
Agglutination Test for RhD
O, O-, A, A-, B, B-, AB, AB-
28
Is PEGylated RhD Immunogenic?
RhD Allorecognition Using RhD- Antigen Presenting
Cell (APC Dendritic-Like Cells) Cultures.
29
Is PEGylated RhD Immunogenic?
RhD Allorecognition RhD- PBMC Proliferation
Following RhD Peptide Presentation By RhD- APC at
48 Hours.
A
B
C
30
Reducing The Flavors of Blood?
Immunocamouflage of RhD Improving blood
utilization and availability.
8 Crucial Flavors Some Realatively Rare...
31
Stealth RBC The Challenges
To Being Famous and Rich
Corporate Considerations It is NOT the next
Viagra Drug Approval Process
32
Immunocamouflage of Blood
What About Other Blood Cells?
33
Paris Hilton School of Medicine IMMUNOLOGY 101
Multiple determinants on cells govern whether
individuals may be potential tissues donors or
tissue recipients.
34
Immunocamouflage Loss of Allorecognition
35
Global Immunocamouflage of Either the APC or
Lymphocyte Prevents Allorecognition Both In Vitro
and In Vivo
CONTROL
36
A Need To Innovate Immunocamouflage of Blood
Cells is Easily and Effectively Accomplished.
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
37
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