blood.ca blood.ca canadian blood services it’s in you to give mark d. scott

BLOOD.CA WWW.BLOOD.CA WWW.BLOOD.CABLOOD.CA WWW.BLOOD.CA WWW.BLOOD.CACanadian Blood Servicesit’s in you to giveMark D. Scott 04/2009 1

Immunocamouflage (Immunocamouflage (PEGylationPEGylation) ) of Red Blood Cellsof Red Blood Cells

The Canadian Blood ServicesThe Canadian Blood ServicesUniversity of British ColumbiaUniversity of British Columbia

Center for Blood Research & Department of Pathology & Laboratory Medicine

The Canadian Blood ServicesThe Canadian Blood ServicesUniversity of British ColumbiaUniversity of British Columbia

Center for Blood Research & Department of Pathology & Laboratory Medicine

Mark D. Scott, Ph.D.Mark D. Scott, Ph.D.Senior Scientist & Clinical ProfessorSenior Scientist & Clinical Professor

Associate Director, Intellectual Property and Business DevelopmentAssociate Director, Intellectual Property and Business Development


Red Blood CellsRed Blood Cells PlateletsPlatelets White Blood CellsWhite Blood Cells CollectionCollection DistributionDistributionSupplySupply

Red Blood CellsRed Blood Cells PlateletsPlatelets White Blood CellsWhite Blood Cells CollectionCollection DistributionDistributionSupplySupply

RBC PROBLEMS:RBC PROBLEMS: Lack of O- Blood, Alloimmunization, Shelf-Life, Cost of

Production, Cost of Distribution

RBC PROBLEMS:RBC PROBLEMS: Lack of O- Blood, Alloimmunization, Shelf-Life, Cost of

Production, Cost of Distribution

Canadian Blood Service


The Holy GrailThe Holy Grail

Don’t need (as many) donors!

No Blood Groups - carefree…!

Long shelf life - months...!

Solving the Problems of Traditional Blood Products

BloodBlood Substitutes!BloodBlood Substitutes!


““Blood SubstitutesBlood Substitutes””

• • PerflurocarbonsPerflurocarbons• • PerflurocarbonsPerflurocarbons1980s-1990s1980s-1990s

• Complement Activation• Vascular Leakage• Other RBC Functions?

• Complement Activation• Vascular Leakage• Other RBC Functions?

XX

• • Purified Purified HemoglobinsHemoglobins

Free & Liposome EncapsulatedFree & Liposome Encapsulated

• • Purified Purified HemoglobinsHemoglobins


1980s-2000s1980s-2000s

What Does The Holy Grail

Look Like?

• Toxicity• Small Size• Vascular Leakage• Tissue Iron Loading• Other RBC Functions?

• Toxicity• Small Size• Vascular Leakage• Tissue Iron Loading• Other RBC Functions?

XX


A Brief History of Hb-Based A Brief History of Hb-Based Blood SubstitutesBlood Substitutes

1980 - 20081980 - 2008

1980s:1980s: US Military BEGINS massive (and expensive) research program to develop hemoglobin (Hb) based blood substitutes. Numerous private companies (e.g., Somatogen, Baxter, HemosolHemosol, Northfield, BioPure) begin commercialization of yeast-produced, human and bovine based Hb-based blood substitutes.

1990s: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:2000s: Despite two decades of commercial (e.g., HemosolHemosol ) 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.


030303 $288.00

090224090224 $0.08$0.08

BioPureBioPure (BPUR)(BPUR)

Hemopure: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: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.

Produces BOVINEBOVINE sourced HemopureHemopure (human

use) and OxyglobinOxyglobin (veterinary use).

Produces BOVINEBOVINE sourced HemopureHemopure (human

use) and OxyglobinOxyglobin (veterinary use).

Cost to transfuse a “YellowYellow” Lab once*:

~$1000.00

Cost to transfuse a “YellowYellow” Lab once*:

~$1000.00

*At the recommended dosage


So Why Does Purified Hemoglobin Fail?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!


In General, In General, “Blood Substitutes”“Blood Substitutes” Have Been Have Been A Major DisappointmentA Major Disappointment

In General, In General, “Blood Substitutes”“Blood Substitutes” Have Been Have Been A Major DisappointmentA Major Disappointment

“Hemoglobin shall be INSIDE red blood cells…” “except, of course,

in earthworms.”

“Hemoglobin shall be INSIDE red blood cells…” “except, of course,

in earthworms.”

The Book of Darwin: Chapter 14, Verse 8

The Book of Darwin: Chapter 14, Verse 8

However, Darwin’s teachings requires However, Darwin’s teachings requires that the that the inherent antigenicity inherent antigenicity and and

immunogenicity immunogenicity of the RBC be tamed.of the RBC be tamed.

However, Darwin’s teachings requires However, Darwin’s teachings requires that the that the inherent antigenicity inherent antigenicity and and

immunogenicity immunogenicity of the RBC be tamed.of the RBC be tamed.

Perhaps DarwinDarwin Has A Point!


““Blood - It’s In You To GiveBlood - It’s In You To Give®®””®®Canadian Blood ServicesCanadian Blood Services

Purified Purified HemoglobinsHemoglobins


Purified Purified HemoglobinsHemoglobins


PerflurocarbonsPerflurocarbonsPerflurocarbonsPerflurocarbons

ImmunocamoulfagedImmunocamoulfaged RBCRBC

Taming the Taming the inherent antigenicityinherent antigenicity and and immunogenicity immunogenicity of the RBC.of the RBC.

Meeting Darwin’s Requirements....Meeting Darwin’s Requirements....


Since the mid-1970’s Since the mid-1970’s methoxypoly(ethylene glycol) methoxypoly(ethylene glycol) [mPEG] [mPEG] has has been “glued” to proteins to prolong circulation and prevent immune been “glued” to proteins to prolong circulation and prevent immune recognition. recognition. PEGylated enzymes are currently used for the clinical PEGylated enzymes are currently used for the clinical

treatment of enzymopathies.treatment of enzymopathies.

Immunocamouflage of CellsImmunocamouflage of Cells

My Laboratory Pioneered The

Immunocamouflage of Intact Cells


BLOOD.CA WWW.BLOOD.CA WWW.BLOOD.CABLOOD.CA WWW.BLOOD.CA WWW.BLOOD.CACanadian Blood Servicesit’s in you to giveMark D. Scott 02/2009

CarbowaxCarbowax®®

In 1927 In 1927 Prestone Prestone introduced introduced Ethylene Ethylene GlycolGlycol** and the era of modern automotive and the era of modern automotive

antifreeze begunantifreeze begun ((thus saving modern Canadathus saving modern Canada).). **replacing such things as molasses, honey and methyl alcohol.replacing such things as molasses, honey and methyl alcohol.

Poly(Poly(ethylene glycolethylene glycol)) is repeating units of is repeating units of

automobile antifreeze!automobile antifreeze!

In 1927 In 1927 Prestone Prestone introduced introduced Ethylene Ethylene GlycolGlycol** and the era of modern automotive and the era of modern automotive

antifreeze begunantifreeze begun ((thus saving modern Canadathus saving modern Canada).). **replacing such things as molasses, honey and methyl alcohol.replacing such things as molasses, honey and methyl alcohol.

Poly(Poly(ethylene glycolethylene glycol)) is repeating units of is repeating units of

automobile antifreeze!automobile antifreeze!

Poly(Poly(Ethylene GlycolEthylene Glycol))Poly(Poly(Ethylene GlycolEthylene Glycol))Carbide and Carbin Chemicals Carbide and Carbin Chemicals Corporation (Union Carbide)Corporation (Union Carbide)

?! That can’t be ?! That can’t be safe!?safe!??! That can’t be ?! That can’t be safe!?safe!?

( )n

C C C OHHO

H

HH

H H H

HH

OC


Is PEG Safe?Is PEG Safe?By chemically linking several By chemically linking several ethylene glycolethylene glycol monomersmonomers

togethertogether,, a a non-toxic polymernon-toxic polymer [ [i.e.,i.e., Poly(ethylene glycol)Poly(ethylene glycol)]] is is produced that is commonly used in produced that is commonly used in modern medicine.modern medicine.

DeadlyDeadlyDeadlyDeadlySafeSafeSafeSafe

SafeSafeSafeSafeAt very HIGH levels, PEG oral toxicityoral toxicity arises due to GI “flow” effects…

Oral Rat LDOral Rat LD5050 for: for: Monomer 4.7 gm/kgPEG 200: 28 gm/kgPEG 1450: 40 gm/kgPEG 4000: >50 gm/kg

Oral Rat LDOral Rat LD5050 for: for: Monomer 4.7 gm/kgPEG 200: 28 gm/kgPEG 1450: 40 gm/kgPEG 4000: >50 gm/kg

~ 4 kg for me...


PEG: PEG: Safe and Non-ToxicSafe and Non-ToxicShaffer, C., Critchfield, F. The absorption and excretion of the solid polyethylene glycols

("Carbowax" compounds). Journal of the American Pharmaceutical Association 36:152-157; 19471947.

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; 19471947.

ORALORAL (10 g bolus):(10 g bolus):

Safe. No measurable absorption from gut, but at high concentrations this industrial “lubricant” did cause diarrhea.

INJECTIONINJECTION (1 g in 20 ml):(1 g in 20 ml):

Safe. ~90% of PEG excreted by kidneys within 12 hours.““Gim’me da Gim’me da carbocarbo or I’ll or I’ll wax wax ya.”ya.”

19471947


Adhesives Agriculture Ceramics

CosmeticsCosmetics and Personal CarePersonal Care Electronics

ElectroplatingElectropolishing

FoodFood Processing

Household Products

Lubricants Metalworking

Paints and Coatings Paper and Paper Products

PharmaceuticalsPharmaceuticals Printing and Inks

RubberElastomers

Textiles Wood Treating

PEGPEGIndustrial-Medical-Consumer ApplicationsIndustrial-Medical-Consumer Applications


How Does PEG WorkHow Does PEG WorkHow Does PEG WorkHow Does PEG WorkPEG is a water-soluble, nontoxic, low immunogenicity polymer readily cleared from the body. Approved for oral, intravenous,

intramuscular, subcutaneous and topical administration.

( )n

C C C OHHO

H

HH

H H H

HH

OC

Physically creating a

semi-dense cloud above

the cell surface


Immunocamouflage of CellsImmunocamouflage of CellsImmunocamouflage of CellsImmunocamouflage of Cells

BLOOD

TISSUES

SNOTSNOT

Hiding the Hiding the “Weapons of Mass Destruction”“Weapons of Mass Destruction” in in TransfusionTransfusion, , TransplantationTransplantation, and , and Family MedicineFamily Medicine

Hiding the Hiding the “Weapons of Mass Destruction”“Weapons of Mass Destruction” in in TransfusionTransfusion, , TransplantationTransplantation, and , and Family MedicineFamily Medicine


RBC Structure, RBC Structure, Function, and In Vivo Function, and In Vivo

Survival?Survival?

Do mPEG-RBC “Work”....



Stealth Erythrocyte Are Stealth Erythrocyte Are NORMALNORMAL......

25

Lysi

sLy

sis

Grafting Process Does NOT Harm The RBC (<1% Lysis)

Normal Human RBC ParametersPEGylated Murine RBC Exhibit Normal In Vivo Survival

or at least appear to be normal...or at least appear to be normal...


Biophysical Effects of Biophysical Effects of mPEG GraftingmPEG Grafting

How Does the grafted mPEG

“Work”....


STEALTH CELLS - Charge CamouflageSTEALTH CELLS - Charge CamouflageSTEALTH CELLS - Charge CamouflageSTEALTH CELLS - Charge Camouflage

Modified from: Scott, M.D. et al. (2000)

Transfusion Medicine Reviews, 14:53-63.

Cell Charge Plays An

Important Role In Immune

Recognition.

Cell Charge Plays An

Important Role In Immune

Recognition.


Cell-Cell Interaction: Erythrocyte SedimentationCell-Cell Interaction: Erythrocyte Sedimentation

Modified From: Bradley, A.J. et al. (2002)

Biochimica et Biophysica Acta, 1561:147-58.

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6

5 mM4 mM3 mM2 mM1.2 mM

0.6 mM

0 mM

Untreated

BTC-mPEG 20 kDa

Hours

Ery

thro

cyte

Sed

imen

tati

on R

ate

(mm

)

RBC Specific Gravity RBC Specific Gravity = = 1.0951.095

Plasma Specific Gravity Plasma Specific Gravity = = 1.0311.031

Rouleaux FormationRouleaux Formation

ControlControl

mPEG-RBCmPEG-RBC

Charge MediatedCharge Mediated


Cell-Protein Interaction: Decreased Fluorescent Fluorescent ProteinProtein Adsorption to Bare or mPEG-Modified

Latex Particles (8 µm).

20 kDa SVA-mPEGBare Latex


Decreased Low Shear Viscosity: Decreased Low Shear Viscosity: Utilization For Vascular Utilization For Vascular Occlusive Diseases (e.g., SCD)?Occlusive Diseases (e.g., SCD)?

Like SyrupLike Syrup

BadBadLike Water Like Water

GoodGood


Immunocamouflage of theImmunocamouflage of the

Rh Blood Group AntigensRh Blood Group AntigensThe Rh antigen family

is located at the membrane surface

does NOT extend very far out.

RhD – making the

“Near” Universal RBC

10 nm

10 nm

20 nm


Mea

n C

ell

Flo

resc

ence

Polymer Grafting Effectively Polymer Grafting Effectively Attenuates Antibody Attenuates Antibody Recognition of RhcRecognition of Rhc

Reducing The Risk Of Alloimmunization Reducing The Risk Of Alloimmunization (Rh Family)(Rh Family)

BLOOD GROUP ANTIGEN Percent Immuno-

genicity¢

Hemolytic

Trans. ReactionPrimary Ab

ABOABO A,BA,B 100100ªª YesYes IgMIgM

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

Single Exposure Risk of AlloimmunizationDerived from “Clinical Experimentation”

2% Risk

??% Risk

¢ Chance of AlloimmunizationFollowing Each Antigen Positive

Transfusion In A Null Individual


Immunocamouflaged of RhDImmunocamouflaged of RhD

Control 0.6 mM

1.2 mM 2.4 mM

5 mM

Agglutination Test for

RhDRhD

O+, O-, A+, A-, B+, B-, AB+, AB-


Is PEGylated RhD Immunogenic?RhD Allorecognition Using RhD- Antigen Presenting Cell (APC;

Dendritic-Like Cells) Cultures.

ImmatureDC-Like

5

Days

IL-4GM-CSF

Plasma

PBMC

Ficoll-Hypaque

RBC

(5hrs)

RhD- Blood

Attach PBMC

RhDPeptide

1 Hour(remove)

2

Days

IL-4GM-CSFIL-1IL-6TNF-αPGE-2

CD83CD86HLA-DR

CD80

MatureDC-Like

Flow Cytometer Analysis

2-4 Days

CFSE-Labeled Fresh Autologous RhD- PBMC

CFSE

Proliferation


Is PEGylated RhD Immunogenic?

RhD Allorecognition: RhD- PBMC Proliferation Following RhD Peptide Presentation By RhD- APC at 48 Hours.

RhD Peptide(Pos. Control)

Proliferation

75%75%

No RhD(Neg. Control)

Proliferation

1.3%1.3%

SVA-mPEG RhD Peptide

Proliferation

0.5%0.5%

A B C


Reducing The “Flavors” of Blood? Reducing The “Flavors” of Blood?

Immunocamouflage of RhD Improving blood utilization and availability.

8 Crucial Flavors8 Crucial FlavorsSome Realatively Rare...

Immunocamouflaged RBCImmunocamouflaged RBC Type O ~ 44% Type A ~ 42%

Type B ~ 10% Type AB ~ 4%


Stealth RBC: Stealth RBC: The ChallengesThe ChallengesTo Being Famous and Rich

CorporateCorporateConsiderationsConsiderations

It is It is NOTNOT the next Viagra the next Viagra

Drug ApprovalDrug ApprovalProcess Process

$$$$

$$$$


Transfusion-Associated Graft Versus Host Disease

32

Immunocamouflage of BloodImmunocamouflage of Blood

What About Other Blood Cells?

BLOOD


Paris HiltonParis Hilton®® School of Medicine: School of Medicine:

IMMUNOLOGY 101IMMUNOLOGY 101Paris HiltonParis Hilton®® School of Medicine: School of Medicine:

IMMUNOLOGY 101IMMUNOLOGY 101Multiple determinants on cells govern whether individuals may be Multiple determinants on cells govern whether individuals may be

potential tissues donors or tissue recipients.potential tissues donors or tissue recipients.

AntigensParis and Not Paris

AntigensParis and Not Paris

PARISPARIS

USUS

Stop


Immunocamouflage:Immunocamouflage:

Loss of AllorecognitionLoss of AllorecognitionImmunocamouflage:Immunocamouflage:

Loss of AllorecognitionLoss of Allorecognition

0

100 101 102 103 104

1000

FSC

-Hei

ght

CD28-FITC

72.88%

27.12%

Control

0

100 101 102 103 104

FSC

-Hei

ght

CD28-FI TC

0.20%

99.80%

10002.4 mM mPEG

per 1 x 107 cells

CD28+CD28+

T Cell APC

CD28 CD80 (B7)

Essential CostimulatoryPathway

3 H-T

hym

idin

e In

corp

orat

ion

(C

PM

)

0

10000

20000

30000

40000

2 .01 .00 0 .5 1 .5 2 . 5

Responder Cell Concentration (x 10Responder Cell Concentration (x 1055))

Unmodified

mPEG-Modified


Global Immunocamouflage of Either the APC or Lymphocyte Prevents Allorecognition

Both In Vitro and In Vivo

CONTROL mPEG-MODIFIED

Allorecognition

Costimulation

AdhesionXXXXXX


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

Potential Applications Potential Applications Include:Include:

A Need To Innovate:A Need To Innovate: ImmunocamouflageImmunocamouflage of Blood Cells is of Blood Cells is Easily and Effectively Accomplished.Easily and Effectively Accomplished.

A Need To Innovate:A Need To Innovate: ImmunocamouflageImmunocamouflage of Blood Cells is of Blood Cells is Easily and Effectively Accomplished.Easily and Effectively Accomplished.

This is not a broken MenorahThis is not a broken Menorah


STUDENTS &POSTDOCSSTUDENTS &POSTDOCSAmanda Bradley, Ph.D.Amanda Bradley, Ph.D.Audrey Chen, Ph.D.Audrey Chen, Ph.D.YevgeniyaLeYevgeniyaLe, Ph.D., Ph.D.Kari Murad, Ph.D.Kari Murad, Ph.D.Troy SuttonTroy Sutton, Ph.D., Ph.D.Dan Wang, Ph.D.Dan Wang, Ph.D.

TECHNICIANSTECHNICIANSNobu Nakane, M.S.Nobu Nakane, M.S.Janet TongJanet TongWendy ToyofukuWendy Toyofuku

COLLABORATORSCOLLABORATORSJohn W. Eaton, Ph.D.John W. Eaton, Ph.D.Elisabeth Maurer, Ph.D.Elisabeth Maurer, Ph.D.Marshal Henri PétainMarshal Henri Pétain

STUDENTS &POSTDOCSSTUDENTS &POSTDOCSAmanda Bradley, Ph.D.Amanda Bradley, Ph.D.Audrey Chen, Ph.D.Audrey Chen, Ph.D.YevgeniyaLeYevgeniyaLe, Ph.D., Ph.D.Kari Murad, Ph.D.Kari Murad, Ph.D.Troy SuttonTroy Sutton, Ph.D., Ph.D.Dan Wang, Ph.D.Dan Wang, Ph.D.

TECHNICIANSTECHNICIANSNobu Nakane, M.S.Nobu Nakane, M.S.Janet TongJanet TongWendy ToyofukuWendy Toyofuku

COLLABORATORSCOLLABORATORSJohn W. Eaton, Ph.D.John W. Eaton, Ph.D.Elisabeth Maurer, Ph.D.Elisabeth Maurer, Ph.D.Marshal Henri PétainMarshal Henri Pétain

Immunocamouflage AcknowledgmentsImmunocamouflage AcknowledgmentsImmunocamouflage AcknowledgmentsImmunocamouflage Acknowledgments

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