us food and drug administration: 2005-4165s1 06

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Division of Hematology

Basil Golding M.D.,

Division Director

Office Site Visit 2005




PIs: 3 2 2 4 Total =

11

LBVB

Biochemistry &

Vascular Biology

LCH

Cellular

Hematology

LH

Hemostasis

LPD

Plasma

Derivatives

CRB

Clinical Review

Branch

Research/Review Units




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 bloodis 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

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-raffinos•Modified cysteines



HBOC-Induced Endothelial Cytotoxicity

MediumRedox Active

ααHb

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



• Establishing methodologies that

distinguish between functional andnon-functional HBOCs

• Development of an endothelium-basedassay that correlates with HBOC

toxicity in vivo

HBOCs: Outcomes

(LBVB)



Alpha-1-Proteinase Inhibitor (α1-PI)

(LBVB)


• Most patients with hereditary α1-PI deficiencydevelop fatal emphysema and about 15%develop severe liver disease.

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



α1-PI: SafetySafety


Aggregated α1-PI can cause adverse

events and decreases potency of α1-PI.

• How do α1-PI polymers form?

• How can polymer formation be avoidedduring manufacture?



reactive

center

loop

freesulfhydryl

A β-sheet

Crystal Structure of α1-PI

flexiblereactive loop

Original Loop-Sheet Model, based on indirect evidence

α 1-PI polymerization isinitiated by partial

unfolding of monomer.



New model explains spontaneous polymerization of dimer

to form aggregate.

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

Dimer Formed By Partial Unfolding of Monomer Continues to

Polymerize After Refolding to Form Aggregate.

High Aggregate

SE-HPLC



α1-PI: Outcomes

• New understanding of α1-PI polymer formation.

• This knowledge can be used to minimizeunfolding of monomers and dimer formationduring manufacturing, e.g. adding appropriatestabilizers during heat treatment .

Future plansInvestigate the conformational change in dimers

that results in spontaneous polymerization.



Immune Globulin Intravenous

(LPD)


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

– Sterile filtration of final product does notremove microbial components, e.g. LPS,

DNA.

• Microbial contaminants proinflammatorycytokines side effects



Immune Globulin Intravenous

Product Contamination


• 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

NFκ -B NF- κ B-

luciferase

Nucleus

http://intimm.oupjournals.org/content/vol17/issue1/images/large/intimmdxh186f01_4c.jpeg














TLR2 TLR4/MD2 TLR90

5

None

PGNEC-LPS

CpG ODN

12.515.0

17.5

20.0

22.5

R L U

HEK-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 detectmicrobial components.

• These cell lines will be used to

develop a rapid high throughputsystem for testing IGIVs and other CBER products for microbialcontaminants.



Cellular Components: Platelets

(LCH)


• 10 million PLT units are transfused annually

in the US.• Transfusion of suboptimal products leads todecreased circulation time more transfusions increased risk of infection and

alloimmunization.




• 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)

A i l M d l f E l ti D t



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 30 p e r c e n t h u m a n p l a t e l e t s i n c i r c u l a t i o

n ( % )

-10

0

10

20

30

40

50

60

70

8090

100

Control (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

p e r c e n t h u m a n p l a t e l e t s i n S C I D m o u s e c i r c u l a t i o n ( % )

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 withadverse events (thrombosis,

inflammation).

Vi l D i d A i i l



Viral Detection and Antiviral

Antibodies: Immune globulins

(LPD) Public Health Impact:

An estimated 2 million Americans suffer fromhepatitis C infection.

• ~ 70% chronic hepatitis

• Sequelaeliver fibrosis cirrhosis HCC



Viral Detection and Antiviral

Antibodies: Immune globulins

(LPD)


• 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-polψ GFP

HCV E1/E2

CMV

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



PseudoparticleHepatitis

Cases

≥1:320≥1:320

≥1:320

≥1:320

<1: 20

<1: 20<1: 20

<1: 20

18

0

42

60

Anti-HCV Screening Removed Nt Abs to

HCV and Compromised Safety of IGIV

Implicated lots made fromanti-HCV screened plasma

in 1993

Non implicated lots made

from anti-HCV unscreenedplasma 1988-1990

IGIV (Not V.I.)

Yu et al, PNAS 2004

0

00



HCIGIV: Outcomes

(LPD)

• HCV neutralization in pseudoparticle assaycorrelates with protection or lack of infectionin chimps.

• Pseudoparticle neutralization assay willfacilitate development of new HCIGIVproducts.

Future Plans• Identification and characterization of

neutralizing epitopes



Inhibitory Antibodies to Factors VIII/IX

(LH)


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 thatcontrol whether patients make

antibodies to Factor VIII/IX

• Developing pre-clinical models to predictproduct efficacy



Mouse Pre-Clinical Models

Using genetically well-characterizedinbred mouse strains we showedthat:

– MHC genes, T-cell receptors, andzinc-α-2-glycoprotein 1 genesinfluence the antibody responseto human factor VIII.

– MHC genes and to a lesser extent,

cytokine genes (IL10, Interferon-γ)control the antibody response tohuman factor IX.Lozier, et al., Blood

2005;105:1029-1035.



Hemophilia A Dog

Pre-Clinical ModelThe 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 tohuman hemophilia A.

The dogs make inhibitors when treatedwith 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 inhumans.

• The Chapel Hill hemophilia A dogs are anideal model for preclinical evaluation of

product potential to induce inhibitoryantibodies.



Immune Globulins:

Counter-Terrorism (LPD)


• Anthrax is a major threat to public health

and security.• Smallpox poses a potential bioterrorism

threat. Widespread vaccination isexpected to cause fatalities in susceptible

individuals – Immune-compromised – Eczema



Immune Globulins:

Counter-Terrorism (LPD)


In vitroand

in vivomodels 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 anthracis

Lethal Factor Lethal Factor Edema Factor Edema Factor AndAnd

Or Or

ProtectiveProtective

AntigenAntigen

(PA)(PA)

Protective AntigenProtective Antigen

(PA) heptamers(PA) heptamers

Cell lysisCytokines

L T

E T Edema

Pil t t di i h



Pilot studies in sheep

Sheep 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 sheepantibodies

DBA/2

Sh D i d A th IG P t t DBA 2 i F

http://www.havanastreet.com/clipart/objects/pages/syringe_gif.htm



Sheep-Derived Anthrax IG Protect DBA-2 mice From

a Lethal Sterne Spore (1x106) Challenge (IP)

10

N u m

b e r o f m i c

e s u r v i v i n g

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

01

2

3

4

5

6

7

8

9

1 2 3 4 5 6 7 8 9 10 11

Control Ab

Anti PA

CiproAnti PA + Cipr

Days after challenge

P i Eff f A h I Gl b li



Protective Effects of Anthrax Immune Globulin

(LBVB)

Time (h)

R e s i s t a n c e ( %

c o n t r o 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


• Complications of smallpox vaccination: – Progressive vaccinia –

• VIG reduces fatality: 100% 50%

– Eczema Vaccinatum• VIG reduces fatality: 30% 3%



VIG Products: Efficacy

(LPD)


• 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:



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

VIGIV Testing in Scarified SCID Mice:



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+10e6

Virus Only 10e5

VIG+10e5

Virus Only 10e6

Days

P e r c e n t S u r v i

v a l

VIGIV Testing in Scarified SCID Mice:

Post-exposure Prophylaxis Efficacy

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

4 long-term

disease-freesurvivors

VIG O t



VIG: Outcomes

(LPD)

• SCID model used to demonstrate that

VIGIV can reduce vaccinia lethality inpre- 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 InternationalWorking 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 α1-PI reference standard

• Identifying/characterizing HCV neutralizingepitopes and enriching HCV neut. Abs

• Assessing neut. Abs to HAV, B19, HBV and

other viral pathogens byin vitro

culturesystems

• 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

us food and drug administration: 2005-4165s1 06

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