Division of Hematology

Basil Golding M.D.,Division Director

Office Site Visit 2005

Division of Hematology

PIs: 3 2 2 4 Total = 11

LBVB

Biochemistry &Vascular Biology

LCH

CellularHematology

LH

Hemostasis

LPD

PlasmaDerivatives

CRB

Clinical ReviewBranch

Research/Review Units

Division of Hematology Scope of Regulation

Products (Biologics, Drugs, Devices)

• Cellular components of blood e.g. platelets• Plasma-derived (Cohn-Oncley Fractionation)• Analogous recombinant e.g Factor VIII

Clinical indications• Bleeding disorders• Shock/hypovolemia• Infectious diseases/immunological deficits• Replacement therapy in congenital or acquired

deficiencies

Research Priorities:Critical Path

• Safety

– Product toxicity (HBOCs)– Contaminants (Microbial)– Viral transmission (HCV)

• Efficacy

– Standards, assays (HBOCs)– Animal models (platelets)

• Counter-Terrorism (anthrax, smallpox)– In vitro assays of potency– Animal models

Hemoglobin-Based O2 Carriers

Public Health Impact:

Oxygen delivery in situations when blood

is not immediately available or acceptable– Trauma (battlefield, rural areas)– Religious reasons– Blood shortages

Hemoglobin-Based O2 Carriers(LBVB)

Regulatory and Scientific Challenges:

• Characterization of HBOC structure-function • Effects of chemical modifications

• Development of pre-clinical models to evaluate HBOC safety

O-R-PolyHbA0 Hallmarks of Functional Abnormality

• Non-sigmoidal oxygen equilibrium curve

• Non-saturating • Non-cooperative (Hill

coefficient = 1.0 vs. 2.5)• pH insensitivity

log PO2

-0.5 0.0 0.5 1.0 1.5 2.0

Sa

tura

tio

n

0.00

0.25

0.50

0.75

1.00

BloodO-R-PolyHbA0

HbA0

Biochemistry (2002)

Biochemistry (2002), Biochemical J. (2004)

O-R-PolyHbA0

Identification of the Origin of Altered Function

(1) Heme Destabilization

(2) Protein Destabilization (locked T state)

O2 O2

O2

O2 O2

O2 O2

Tense (T) Deoxy

Tense (T) Oxy

Locked (T) State

O2 O2

O2 O2

Relaxed (R) Oxy

Normal Conformational Change

Tetragonal Heme Fe

HbA0

Rhombic Heme Fe

O-R-PolyHbA0

(EPR)

MALDI-MS

O-R-PolyHbA0:Actual Chemical Modification

Boykins,Buehler, Alayash. Proteins (2005)

•Non-specific cross-link•Non-uniform O-raffinose•Modified cysteines

HBOC-Induced Endothelial Cytotoxicity

MediumRedox Active

Hb

D’Agnillo, Am J Physiol. (2004), Blood (2001)

• Establishing methodologies that distinguish between functional and non-functional HBOCs

• Development of an endothelium-based assay that correlates with HBOC toxicity in vivo

HBOCs: Outcomes(LBVB)

Alpha-1-Proteinase Inhibitor (1-PI)(LBVB)

Public Health Impact:

• Most patients with hereditary 1-PI deficiency develop fatal emphysema and about 15% develop severe liver disease.

• Such deficient patients (approx. 4,000) can benefit from augmentation therapy.

1-PI: Safety Safety

Regulatory and Scientific Challenges:

Aggregated 1-PI can cause adverseevents and decreases potency of 1-PI.

• How do 1-PI polymers form?

• How can polymer formation be avoided during manufacture?

reactivecenterloop

freesulfhydryl

A -sheet

Crystal Structure of 1-PI

flexible reactive loop

Original Loop-Sheet Model, based on indirect evidence

-PI polymerization is

initiated by partial

unfolding of monomer.

New model explains spontaneous polymerization of dimerto form aggregate.

Marszal, Danino, and Shrake: J. Biol. Chem. (2003)

Dimer Formed By Partial Unfolding of Monomer Continues toPolymerize After Refolding to Form Aggregate.

High Aggregate

SE-HPLC

1-PI: Outcomes

• New understanding of 1-PI polymer formation.

• This knowledge can be used to minimize unfolding of monomers and dimer formation during manufacturing, e.g. adding appropriate stabilizers during heat treatment .

Future plansInvestigate the conformational change in dimers that results in spontaneous polymerization.

Immune Globulin Intravenous(LPD)

Public Health Impact:

Product Contamination • Infusion of IGIV causes adverse events – 25%

– Sterile filtration of final product does not remove microbial components, e.g. LPS, DNA.

• Microbial contaminants proinflammatory cytokines side effects

Immune Globulin Intravenous Product Contamination

Regulatory and Scientific Challenges:

• Need for rapid high throughput assays to detect microbial components

• Adapt cell lines expressing Toll-like receptors as a detector system

Nucleus

Toll-Like Receptors (TLRs) and Their Ligands

Takeda and Akira Inf. & Imm. 2005

Pathogen Recognition

NF-BNF-B-luciferase

Nucleus

TLR2 TLR4/MD2 TLR90

5

NonePGNEC-LPSCpG ODN

12.515.017.520.022.5

RL

UHEK-293 Cells Transfected with TLRs and a

Reporter Gene Respond to Microbial Ligands

Huang et al, J. Immunol. 171(3):1441-6. 2003

IGIV: Outcomes

• Cell lines expressing multiple TLRs and a reporter gene can detect microbial components.

• These cell lines will be used to develop a rapid high throughput system for testing IGIVs and other CBER products for microbial contaminants.

Cellular Components: Platelets(LCH)

Public Health Impact:

• 10 million PLT units are transfused annuallyin the US.

• Transfusion of suboptimal products leads to decreased circulation time more transfusions increased risk of infection and

alloimmunization.

Regulatory and Scientific Challenges:

• Testing for Efficacy• In vitro tests - informative but not predictive• In vivo survival of radio-labelled platelets in

humans is burdensome• Adverse Effects

• Possibly due to microparticles

Cellular Components: Platelets(LCH)

Animal Model for Evaluating Damage to Human Platelets During Collection,

Processing and Storage

Survival of human platelets in Control and SCID mice

0 5 10 15 20 25 30perc

ent

hum

an p

late

lets

in c

ircul

atio

n (%

)

-10

0

10

20

30

40

50

60

70

80

90

100Control (FVB) MiceSCID Mice

* p < .03

p < 0.34

p < 0.12

Control SCID

In vivo performance of 1 and 7 day old human platelets

in SCID mice

Time (hours)

0 1 2 3 4 5

perc

ent h

uman

pla

tele

ts in

SC

ID m

ouse

circ

ulat

ion

(%)

0

20

40

60

80

100

* p < 0.001 * p < 0.001Day 1, Filled SymbolsDay 7, Open Symbols

1 day 7 day

Platelets: Outcomes(LCH)

• New in vivo assay in SCID mice was developed for measuring platelet survival.

• Flow cytometric assays were developed to detect microparticles in platelet products: this will enable us to determine whether MPs in products are associated with adverse events (thrombosis, inflammation).

Viral Detection and Antiviral Antibodies: Immune globulins

(LPD)

Public Health Impact:

An estimated 2 million Americans suffer from

hepatitis C infection.

• ~ 70% chronic hepatitis

• Sequelae

liver fibrosis cirrhosis HCC

Viral Detection and Antiviral Antibodies: Immune globulins

(LPD)

Regulatory and Scientific Challenges:

• Screening plasma for HCV

• Measuring HCV neutralizing Abs in HCIGIV

– no in vitro system or small animal model is available for HCV infectivity

– chimpanzee is the only model

HCV Pseudoparticle System

Step 3: Quantification of infection by FACS

Step 1: Transfection and particle production

CMV

gag-pol GFP

HCV E1/E2CMV

Step 2: Infection of target cells

HCVpp entryGFP

expression

293 T cell(s)

Huh-7 cells

Pseudoparticle

<1 : 20

1 : 320

1 : 320

Sample

Anti-HCV

neg pos

pos

Control IGIV

HCIGIV

HCIGIV(HCV RNA+)

Chimpanzee

Protected

Not infectious

Not protected

V.I.

S/D

S/D

None

Pseudoparticle Assay Correlates With In Vivo Chimp Data

Yu et al, PNAS 2004

Pseudoparticle

Hepatitis Cases

1:320

1:320

1:320

1:320

<1: 20

<1: 20

<1: 20<1: 20

18

0

4 2

60

Anti-HCV Screening Removed Nt Abs to HCV and Compromised Safety of IGIV

Implicated lots made from anti-HCV screened plasma in 1993

Non implicated lots made from anti-HCV unscreened plasma 1988-1990

IGIV (Not V.I.)

Yu et al, PNAS 2004

0 0

0

HCIGIV: Outcomes (LPD)

• HCV neutralization in pseudoparticle assay correlates with protection or lack of infection in chimps.

• Pseudoparticle neutralization assay will

facilitate development of new HCIGIV products.

Future Plans

• Identification and characterization of neutralizing epitopes

Inhibitory Antibodies to Factors VIII/IX (LH)

Public Health Impact:

Neutralizing antibodies to coagulation factors complicate the use of Factor VIII in ~20% of patients with severe hemophilia.

Inhibitory Antibodies to Factors VIII/IX (LH)

Research and Regulatory Challenges:

• Understanding the genetic factors that control whether patients make antibodies to Factor VIII/IX

• Developing pre-clinical models to predict product efficacy

Mouse Pre-Clinical Models

Using genetically well-characterized inbred mouse strains we showed that: – MHC genes, T-cell receptors, and

zinc--2-glycoprotein 1 genes influence the antibody response to human factor VIII.

– MHC genes and to a lesser extent, cytokine genes (IL10, Interferon-) control the antibody response to human factor IX.Lozier, et al., Blood

2005;105:1029-1035.

Hemophilia A Dog Pre-Clinical Model

The Chapel Hill hemophilia A dogs have a genetic defect identical to that in ~40% of humans with severe hemophilia A.

The bleeding phenotype is identical to human hemophilia A.

The dogs make inhibitors when treated with dog factor VIII.

Lozier et al, PNAS 2002;99:12991-12996

Factors VIII/IX: Outcomes (LH)

• Mouse genetic studies provide clues for probing the genetic predisposition to induction of inhibitory antibodies in humans.

• The Chapel Hill hemophilia A dogs are an ideal model for preclinical evaluation of product potential to induce inhibitory antibodies.

Immune Globulins:Counter-Terrorism (LPD)

Public Health Impact:

• Anthrax is a major threat to public health and security.

• Smallpox poses a potential bioterrorism threat. Widespread vaccination is expected to cause fatalities in susceptible individuals– Immune-compromised– Eczema

Immune Globulins:Counter-Terrorism (LPD)

Regulatory and Scientific Challenges:

In vitro and in vivo models are required toassess efficacy of:

• Anthrax Immune Globulins

• Vaccinia Immune Globulins

Anthrax Immune Globulins

• Inhalational anthrax– In 2001, 5/11 patients died despite

antibiotic treatment.

• Combined antibiotics and anti-anthrax antibodies may improve survival– Antibiotic targets the bacillus.– Antibodies target the toxins.

Toxins of Bacillus anthracisToxins of Bacillus anthracis

Lethal FactorLethal FactorEdema FactorEdema FactorAnd And Or Or

Protective Protective Antigen Antigen (PA)(PA)

Protective Antigen Protective Antigen (PA) heptamers(PA) heptamers

Cell lysisCytokines

LT

ETEdema

Pilot studies in sheepSheep were immunized with various proteins (PA or LF in

adjuvant) or the Sterne strain agricultural vaccine.

(FEMS Immunology and Med Micro, 2003)

Various anthrax immunogens

Purified sheep antibodies

DBA/2

Sheep-Derived Anthrax IG Protect DBA-2 mice From a Lethal Sterne Spore (1x106) Challenge (IP)

10

Nu

mb

er o

f m

ice

surv

ivin

g

Antibodies (25 mg/kg) given day of challenge and Cipro (10mg/kg) given day after challenge-both given daily

0123456789

1 2 3 4 5 6 7 8 9 10 11

Control Ab

Anti PA

Cipro

Anti PA + Cipro

Days after challenge

Protective Effects of Anthrax Immune Globulin (LBVB)

Time (h)

Re

sis

tan

ce (

% c

on

tro

l)

Medium LT + AIGLT

MEK1 Cleavage Assay

0

20

40

60

80

100

120

0 20 40 60 80

Medium

LT

LT + AIG

Anthrax Immune Globulins: Outcomes (LPD/LBVB)

• “Proof of Concept” that polyclonal antibodies made in animals can protect against anthrax toxins

• Established in-house in vitro and in vivo assays for testing efficacy of Anthrax Immune Globulin products

Vaccinia Immune Globulins

Public Health Impact:

• Complications of smallpox vaccination:– Progressive vaccinia –

• VIG reduces fatality: 100% 50% – Eczema Vaccinatum

• VIG reduces fatality: 30% 3%

VIG Products: Efficacy(LPD)

Regulatory and Scientific Challenges: • How can efficacy and potency for VIG

products be assessed?

• Need for an animal model of severe vaccinia in an immunodeficient host

Day 7

Day 21 Day 28

Day 14

SCID Mouse Model of Progressive Vaccinia:Similarities to Human Disease

• Mimics human route of exposure

• Non-healing primary lesion

• Systemic spread of virus

• Lethality

SCID Mouse Dryvax Scarification at 106, 105 with 10mg X 4 VIG Treatment

0 10 20 30 40 50 60 70 80 90 100 110 120 1300

50

100

VIG+10e6Virus Only 10e5

VIG+10e5

Virus Only 10e6

Days

Per

cen

t S

urv

ival

VIGIV Testing in Scarified SCID Mice:Post-exposure Prophylaxis Efficacy

Vaccinia day 0 - VIGIV day 2, 5, 10, 15

4 long-termdisease-freesurvivors

VIG: Outcomes(LPD)

• SCID model used to demonstrate that VIGIV can reduce vaccinia lethality in pre- and post-exposure treatment.

• SCID model adopted by industry in support of licensure.

• 1st International Standard for von Willebrand Factor Concentrate

• Mega 2/EP BRP Batch 3 International Working Standard for FVIII Concentrates

• 7th International Standard for Factor VIII Concentrate

• 2nd International Standard/FDA Standard Lot K for Thrombin

Reference Standards Established Through International Collaborations (I)

Reference Standards Established Through International Collaborations (II)

• 2nd International Standard/EP BRP1/CBER Lot 4 for Potency of Anti-D Immunoglobulins

• 1st International Standard/CBER Standard for Parvovirus B19 NAT Assays

• 1st International Standard/CBER Standard for Hepatitis A Virus NAT Assays

• 1st International/CBER Reference Reagents to Limit the Anti-D Levels in IGIV Products

DH: Future Directions (I)

• Safety• Studying the association of IGIV on pro-

inflammatory cytokine responses. • Developing NAT and infectivity assays to

determine and quantify HCV and B19 viral variants in plasma-derived products.

• HBOCs: pre-clinical models to evaluate oxidative stress and vasoactivity.

DH: Future Directions (II)

• Efficacy• Establishing WHO 1-PI reference standard

• Identifying/characterizing HCV neutralizing epitopes and enriching HCV neut. Abs

• Assessing neut. Abs to HAV, B19, HBV and other viral pathogens by in vitro culture systems

• VWF: novel assay for evaluation of activity

DH: Future Directions (III)

• Counter-terrorism• Anthrax: Human antibodies from trans-

chromosomal cows will be tested for protective antibodies by in vitro neutralization of toxin (macrophages, endothelial cells) and in mouse models.

• Vaccinia: develop a mouse model for eczema vaccinatum