Gene Transfer and Immunogenicity

BranchSite Visit March 2,

2007 Eda T. Bloom, PhD, Chief Andrew P. Byrnes, PhD, Senior Staff Fellow Suzanne L. Epstein, PhD, Senior Investigator Nancy S. Markovitz PhD, Senior Staff Fellow Carolyn A. Wilson, PhD, Senior Investigator

Takele Argaw, DVM, Staff Fellow

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Problems in Development of Cell and Gene Therapy

Products Lack of preclinical models to predict performance of gene and cell therapy products in vivo

Potential for transmission of infectious agents to the patient and beyond Products often intended for lengthy or

permanent presence in the recipient Complicated manufacture and structure of

products Challenging product characterization and

testing

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The Challenges for GTIB Product safety is affected by virus-

containing products Unintended replicating viruses in viral vectors,

xenotransplantation, and other products Public health concern of spread beyond the patient

Viral vectors carry inherent risks (e.g., toxicity, tumorigenicity, off-target effects)

Immunogenicity impacts safety and efficacy Immune responses to viruses and transgene

products, xenotransplantation and cell therapy products

Immune activity of certain immunotherapy products

GTIB Addresses Challenges Through Critical Path

Research Multiple viral systems, e.g., adenovirus,

filovirus, influenza virus, herpesvirus, retrovirus

Flexible systems to address immunogenicity and function of many OCTGT products

Predictability comes from understanding: Fate and effect in vivo Role of structure and function in safety Interaction with immune system

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Summary of Individual Programs Presented at the March 2 Site Visit

andRegulatory Impact

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Immunobiology of Cellular Therapy and

Xenotransplantation Products: Immunogenicity Issues (Bloom)

•Immunity includes NK cells and T cells, and Ab•Regulatory T cells inhibit CD4+ T cell response

Autologous cellular therapy – cells themselves may be intended to be immunologically active, e.g., NK or T cell immunotherapy

Primarily T cell, and sometimes NK cell immunity (e.g., hES cells & progeny), Ab

xenotransplantation

allotransplantation

autologous

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Expanded baboon CD4+CD25+ T reg cells strongly suppress proliferation and cytokine

production of baboon CD4+CD25- effector cells

To understand role of Tregs in

xenotransplantation in vivo: A preclinical model

will be important

Impact on Cell Therapy and Xenotransplantation

Products NK cells must be considered as immune

mediators for rejection of both xenogeneic transplants and certain allogeneic cell therapy products

Efficacy may be affected by Type(s) of immune response(s) of recipient Microenvironment in patient

Studies of patient immune responses in vitro may predict immunity in vivo Could serve as biomarkers of clinical benefit

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Safety and biodistribution of adenovirus vectors (Byrnes)

>80 active INDs in US for adenovirus vectors>80 active INDs in US for adenovirus vectors Poor pharmacokinetics after systemic injectionPoor pharmacokinetics after systemic injection

Rapid clearance of Ad by the liverRapid clearance of Ad by the liver Inefficient therapy, toxicityInefficient therapy, toxicity

Kupffercells

Control liver After i.v. injection of Ad vector

Ad vectors are taken up by Kupffer cells, which then die

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How do Kupffer cells recognize How do Kupffer cells recognize adenovirus so well?adenovirus so well?

New assay for New assay for quantitating KC quantitating KC uptake of Aduptake of Ad

Scavenger receptors Scavenger receptors are important for KC are important for KC uptake of Aduptake of Ad

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Critical Path issues in adenoviral gene therapy

Kupffer cells prevent Ad gene therapy Kupffer cells prevent Ad gene therapy from reaching its full potentialfrom reaching its full potential

Goals:Goals: Understand how Kupffer cells recognize Understand how Kupffer cells recognize

adenovirusadenovirus Rationally develop strategies to block Rationally develop strategies to block

Kupffer cells in order to enhance gene Kupffer cells in order to enhance gene delivery and decrease vector toxicitydelivery and decrease vector toxicity

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Influenza, the public health problem (Epstein)

High mortality from seasonal outbreaks, concern about pandemic (subtype new to humans). Vaccine supply delayed or inadequate.

Work in this program: New vaccine technologies can cross-protect broadly against divergent influenza A subtypes. Historical data suggest possible cross-protection in humans during the pandemic of 1957.

Maintaining the cold chain Producing vaccines

1313Morbidity also reduced (body weight)

0

1

2

3

4

5

6

7

Lung - Day 5

Log1

0 E

ID50

/ml

A/NP

A/M2

B/NP* *

*P = 0.004

ANOVA

Challenge with A/Thailand/SP83/0417 days after boost

M2 can protect against a lethal H5N1

challengeDNA+rAd

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Implications for public health

and product regulation Addresses DHHS and center-wide

priorities on: Control of epidemic and pandemic influenza. Counter-bioterrorism: control of emerging

infectious disease without having to know which strain is coming

Safety and efficacy impact in gene therapy: antibody and T cell responses to viral vectors can block efficacy, cause immunopathology.

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Identity and Safety Studies of Herpes Simplex Viruses and

Vectors(Markovitz)

Identity/StructureReplicating viruses used in cancer therapy studies contain unreported mutations.

SafetyIn contrast to what is frequently reported, HSVs similar to those in clinical trials do replicate in normal brain cells in mice.

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• R3617 is the parent strain of virus G207, used in clinical trials.

•Sequence analysis proved that the mobility shift was due to a single base substitution

•Resulted in the truncation of the UL3 protein.

Unexpected Mutations in Herpes Simplex Virus for

Clinical Use

MW HSV-1(F

)

45

31

R3617

1 2

UL3C]UL3 WT [

Dambach et al. (2006) Molecular Therapy 13(5): 892-899.

1717

Filoviridae Ebolavirus

Marburg virus

ZaireIvory CoastSudanReston

High fatality rate; rapid disease High fatality rate; rapid disease progression.progression.

No proven cure or vaccineNo proven cure or vaccine Infection suppresses both the innate Infection suppresses both the innate

and adaptive immune responsesand adaptive immune responses Exacerbates pathogenic outcomeExacerbates pathogenic outcome Prevents development of protective Prevents development of protective

immune responseimmune responseDHHS high Priority Bioterrorism Agents

(Wilson)

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Zaire

Ivory Coast

Sudan

VSV-GVSV-G

Anti-F88 MAbs Neutralize Three Ebolavirus Species*

*Shown here with retroviral vector pseudotypes, now extended to WT Ebolavirus

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Impact on Counter-Bioterrorism:Availability of Antibodies for Passive

Transfer

Targeting conserved epitopes: Block Entry → Reduce virus burden Critical for Entry → Avoid potential

for immune escape mutants Broad protection to Filovirus sp.Complements vaccine strategy: Provides immediate protection in

the event of an outbreak (natural or BT)

2020

National Toxicology Program (NTP):

Safety assessment of retroviral vectors for risk of tumorigenicity

Determine the sensitivity of a preclinical model for detecting retroviral vector-mediated insertional tumorigenesis

Impact of vector backbone, dose, and enhancer deletion on tumor frequency Will assess retroviral vectors representing

three different retroviral genera: Gammaretrovirus (MLV) Lentivirus (HIV) Spumavirus (HFV)

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Xenotransplantation: Identification of Porcine Endogenous Retrovirus

Determinants Critical for Human Cell Tropism(Argaw)

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Regulatory Impact of OCTGT Retrovirus Research

Addressing Risks Associated with Retroviral Vectors: Identification of model to assess insertional

mutagenesis from vector Using model to assess relative risks, based on

different vector types, dose, and structure Porcine Xenotransplantation Products

Identification of residues required for transmission of porcine endogenous retrovirus may allow development of means to block infection and reduce risk of transmission

Summary - GTIB Research: Addressing Regulatory

Challenges Gene therapy vector safetyGene therapy vector safety Xenotransplantation safetyXenotransplantation safety Cellular therapy and Cellular therapy and

xenotransplantation product xenotransplantation product efficacyefficacy

Center-wide and Departmental Center-wide and Departmental prioritiespriorities Pandemic influenzaPandemic influenza Counter terrorismCounter terrorism