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Division of Hematology

Basil Golding M.D.,
Division Director

Office Site Visit 2005
Division of Hematology

Research/Review Units

LBVB LCH LH LPD CRB
Biochemistry & Cellular Hemostasis Plasma Clinical Review
Vascular Biology Hematology Derivatives Branch

PIs: 3 2 2 4 Total =
11
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
1.00 HbA0 equilibrium curve
Blood
Saturation

0.75 • Non-saturating
O-R-PolyHbA0
0.50 • Non-cooperative (Hill
0.25 coefficient = 1.0 vs. 2.5)
0.00
• pH insensitivity
-0.5 0.0 0.5 1.0 1.5 2.0

log PO2

Biochemistry (2002)
O-R-PolyHbA0
Identification of the Origin of Altered Function
Tetragonal Heme Fe Rhombic Heme Fe

(1) Heme
Destabilization
O-R-
(EPR) HbA0
PolyHbA0

O2 O2
O2 O2
O2 Locked (T) State

(2) Protein
Destabilization Tense (T) Oxy

(locked T state) O2 O2
O2 O2 Normal Conformational
Change
MALDI-MS Tense (T) Deoxy
O2 O2

Relaxed (R) Oxy
Biochemistry (2002), Biochemical J. (2004)
O-R-PolyHbA0:
Actual Chemical Modification

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

Boykins,Buehler, Alayash. Proteins (2005)
HBOC-Induced Endothelial Cytotoxicity
Redox Active
Medium ααHb

D’Agnillo, Am J Physiol. (2004), Blood (2001)
HBOCs: Outcomes
(LBVB)

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

• Development of an endothelium-based
assay that correlates with HBOC
toxicity in vivo
Alpha-1-Proteinase Inhibitor (α 1-PI)
(LBVB)

Public Health Impact:
• Most patients with hereditary α 1-PI deficiency
develop fatal emphysema and about 15%
develop severe liver disease.
• Such deficient patients (approx. 4,000) can
benefit from augmentation therapy.
α 1-PI: Safety

Regulatory and Scientific Challenges:
Aggregated α 1-PI can cause adverse
events and decreases potency of α 1-PI.

• How do α 1-PI polymers form?
• How can polymer formation be avoided
during manufacture?
Crystal Structure of α 1-PI
A β-sheet
flexible
reactive
reactive loop
center
loop

α 1 -PI polymerization is
initiated by partial
unfolding of monomer.

free
sulfhydryl

Original Loop-Sheet Model, based on indirect evidence
Dimer Formed By Partial Unfolding of Monomer Continues to
Polymerize After Refolding to Form Aggregate.

High Aggregate

SE-HPLC

New model explains spontaneous polymerization of dimer
to form aggregate.

Marszal, Danino, and Shrake: J. Biol. Chem. (2003)
α 1-PI: Outcomes

• New understanding of α 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 plans
Investigate 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
Toll-Like Receptors (TLRs) and Their Ligands

Pathogen
Recognition

NF- κB-
NFκ-B
luciferase

Nucleus
Nucleus

Takeda and Akira Inf. & Imm. 2005
HEK-293 Cells Transfected with TLRs and a
Reporter Gene Respond to Microbial Ligands

22.5
20.0
17.5 None
15.0 PGN
12.5 EC-LPS
RLU

CpG ODN
5

0
TLR2 TLR4/MD2 TLR9

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 annually
in the US.
• Transfusion of suboptimal products leads to
decreased circulation time  more transfusions
 increased risk of infection and
alloimmunization.
Cellular Components: Platelets
(LCH)

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
Animal Model for Evaluating Damage to
Human Platelets During Collection,
Processing and Storage
In vivo performance of
Survival of human platelets in 1 and 7 day old human platelets
Control and SCID mice in SCID mice

percent human platelets in SCID mouse circulation (%)
percent human platelets in circulation (%)

100
90 Control (FVB) Mice
SCID Mice 100
80
70
80
60
50
60
40
p < 0.12
30
20 40

10
* p < .03
p < 0.34
0 20
-10
Day 1, Filled Symbols * p < 0.001 * p < 0.001
0 5 10 15 20 25 30 0 Day 7, Open Symbols

0 1 2 3 4 5
Time (hours)

Control SCID
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 1: Transfection and particle production

CMV HCV E1/E2
CMV gag-pol
ψ GFP

293 T cell(s)
Step 2: Infection of target cells
GFP
HCVpp entry expression

Huh-7 cells

Step 3: Quantification of infection by FACS
Pseudoparticle Assay Correlates
With In Vivo Chimp Data

Sample Anti-HCV V.I. Chimpanzee Pseudoparticle

Control IGIV neg S/D Not protected <1 : 20

HCIGIV pos S/D Protected ≥1 : 320

HCIGIV pos None Not infectious ≥1 : 320
(HCV RNA+)

Yu et al, PNAS 2004
Anti-HCV Screening Removed Nt Abs to
HCV and Compromised Safety of IGIV

IGIV (Not V.I.) Hepatitis
Cases Pseudoparticle

Non implicated lots made 0 ≥1:320
from anti-HCV unscreened 0 ≥1:320
plasma 1988-1990
0 ≥1:320
0 ≥1:320

Implicated lots made from 4 <1: 20
anti-HCV screened plasma 2 <1: 20
in 1993 60 <1: 20
18 <1: 20

Yu et al, PNAS 2004
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-α-2-glycoprotein 1 genes
influence the antibody response
to human factor VIII.
– MHC genes and to a lesser extent,
cytokine genes (IL10, Interferon-γ)
control the antibody response to
Lozier, et al., Blood human factor IX.
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 to
assess 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 anthracis

And
Edema Factor Or Lethal Factor

Protective Protective Antigen
Antigen (PA) heptamers
(PA)
Cell lysis
Cytokines
LT

ET
Edema
Pilot studies in sheep
Sheep were immunized with various proteins (PA or LF in
adjuvant) or the Sterne strain agricultural vaccine.

Various anthrax immunogens

Purified sheep
DBA/2
antibodies

(FEMS Immunology and Med Micro, 2003)
Sheep-Derived Anthrax IG Protect DBA-2 mice From
a Lethal Sterne Spore (1x106) Challenge (IP)
Number of mice surviving

10
9
8 Control Ab
7 Anti PA
6 Cipro
5
Anti PA + Cipro
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
Days after challenge
Antibodies (25 mg/kg) given day of challenge and Cipro (10mg/kg) given day after challenge-both
given daily
Protective Effects of Anthrax Immune Globulin
(LBVB)
120
Resistance (% control)

100
MEK1 Cleavage Assay
80

60

Medium
40 Medium LT LT + AIG
LT
20
LT + AIG
0
0 20 40 60 80

Time (h)
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
SCID Mouse Model of Progressive Vaccinia:
Similarities to Human Disease

Day 7 Day 14
• Mimics human route of
exposure
• Non-healing primary
lesion
• Systemic spread of
virus
Day 21 Day 28
• Lethality
VIGIV Testing in Scarified SCID Mice:
Post-exposure Prophylaxis Efficacy
SCID Mouse Dryvax Scarification at 106, 105 with 10mg X 4 VIG Treatment

100 Virus Only 10e6
Virus Only 10e5
VIG+10e6
VIG+10e5
Percent Survival

50

4 long-term
disease-free
survivors
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Days

Vaccinia day 0 - VIGIV day 2, 5, 10, 15
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.
Reference Standards Established Through
International Collaborations (I)

• 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 (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 α 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