Transcript Document

BBI Biotech Research
Laboratories
Core B
Janet L Lathey, Ph.D.
Director Virology/Immunology
Boston Biomedica, Inc.
Overview
• Founded in 1986
• Leading supplier of innovative products and services to the
life sciences industry
• Approximately 200 employees with facilities in
Massachusetts and Maryland
• Customers include:
– Diagnostics and pharmaceutical manufacturers
– Government research and regulatory agencies
– Proficiency organizations
– End-users of diagnostic test kits
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Biotech Overview
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The research and development arm of the Company for Molecular Biology,
Virology and Immunology
Experienced Scientific Staff consisting of 9 PhD’s and more than 50 scientists
Provides a variety of products and services to BBI operating units and other
outside customers
– Specialty reagents and molecular and cellular biology services
– Blood and tissue processing and repository services
– Clinical trials for domestic and foreign test kit and device manufacturers
Services typically provided under multi-year contracts
Services focused in advanced biomedical research areas
Gaithersburg, Maryland
Frederick, Maryland
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Repository Services
• Study design support for research
and clinical trials
• Training and documentation for
collection sites
• Blood fractionation, PBMC Isolation
and Cryopreservation
• Specimen accessioning utilizing bar
coding and real-time database
• Sample storage and distribution
• Shipping guidance and support
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Molecular Biology Services
• Extraction of Nucleic Acids
– Blood, buccal swabs, tissues
• HIV Drug Resistance analysis
– Viroseq kits from ABI
• Viral Sequencing and
Subtyping
• Viral Load Assays
– Roche COBAS
– Roche Amplicor Monitor
– RT TaqMan PCR
• Recombinant DNA and Library
Screening and DNA Finger
Printing
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Virology/Immunology
• Viral Culture, Titration
and Inactivation
• HIV Drug Susceptibility
Assays
• EBV Transformation
• ELISpot Assays
• Apoptosis Assays
Virology Services and Molecular Biology
Available for Clinical Trials of Anti-retrovirals
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HIV Antigen Detection and Quantitation
HIV Culture, Isolation, and Tropism
Titration of Infectious Virus
Drug Susceptibility Assays
Viral Nucleic Acid Isolation
Viral Nucleic Acid Quantitation
Viral Sequencing
Drug Resistance Testing (Genotyping)
HIV-I Subtypes Collection
Subtype
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A
AG
B
C
D
AE
F
G
H
Group O
HIV-2
# Isolates
3
3
10
7
3
10
5
2
1
4
1
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Our collection includes all members
of the NED (NIH-ENVA-DOD) HIV-I
Subtypes Reference and Standards
Panel
All NED Panel isolates were
sequenced by the VQA to ensure
>98% homology to Panel Seed
Stocks.
VQA EM Particle Counts for most
isolates
RNA concentration >10E+08
copies/mL for all isolates
GenBank Accession Numbers
available
Cultured under GMP conditions
Drug Resistant Isolates
Isolates
139-1
HIV174V/MT2
Biotype
Resistance
References
SI
(R5X4)
AZT
1989 Larder BA, Science 246:
1155
X4
ddI,ddC
xxxHIV-1
LAI-M184V
N119
J 302
3TC
SI (X4)
1993 Schinaizi RF,
Antimicrob Agents Chemother
37:875
nevirapine
saquinavir
1995 Jacobsen H. Virol
206:527
HIV Infectivity Assay
Make 10-fold Dilutions of Virus 10-1 – 10-8
Add Cells (Cell line or PBMC)
Incubate Overnight
Centrifuge Cells and Wash 3 Times
Incubate for 7-28 Days
Feed every 3 to 4 Days
Harvest at Day 7,14, 21 and 28
Measure HIV-1 p24 Antigen
by ELISA
Drug Susceptibility Assays
• Viral isolate is titrated under conditions of
susceptibility assay.
• Decreasing concentrations of drug are diluted with
200 TCID50 of virus and 200,000 PBMC per well
• On day 4 drug is replenished
• On day 7 supernatant is harvested for p24 antigen
assay.
• IC50 is determined based on reduction in p24
production compared to no drug
Drug Family
Alkaloids
Terpenoids
Lignans
Glycosides
Drug
Identification
IC50 (µg/ml)
EC50 (µg/ml)
Therapeutic
Index
ZS-114
>100e
0.8
>125
ZS-147
>100
1.4
>69.9
ZS-156
>100
0.8
>125
IC-2
16.9
0.02
845
IC-9522
10.5
0.0006
16,418
SXZ-LN-3
>100
1.1
>91
SXZ-EL-6
29.9
0.3
70.2
SXZ-EL-12
1.8
0.0006
300
ZS-3
>100
4.2
>23.8
ZS-183
>100
1.0
>100
ZS-188
5.1
1.0
5.0
Anti-retroviral selected with BBI
screening is in Clinical Trials
• Over 5000 plant extracts and derivatives
were screened by BBI Biotech and described
in over 30 publications.
• One drug from the terpenoid group was
selected for further testing.
• A Phase I Clinical Trial has been completed
with this maturation inhibitor, PA 457.
• Panacos expects to start a Phase II trial by
the end of the year.
Specific Aim (1) Screening potential inhibitors: SP antagonists with potential
anti-viral activity will be tested for inhibitory activity against HIV BaL and NL43 propagated in PBMC. Cytotoxicity against PBMC will be determined to
obtain an in vitro therapeutic index (TI).
Compounds with High TI will advance
to the next screening level
Project 2
Test effect on monocyte
cellular function
Project 1
Testing antiviral activity
In other cell types
Remain in Core B
To be tested against
Primary isolates
Specific Aim (2) Efficacy against primary HIV-1 isolates: Characterize the breadth
of inhibitory activity of the compounds against a panel of primary HIV-1
isolates propagated in PBMC.
B and non-B subtypes
Anti-retroviral resistant
T cell- and monocyte-tropic
Isolates isolated by Project 1
In presence or absence of inhibitors
All data is transferred to Core C with screening data from projects 1 and 2.
Complied data will be used to determine best dose of chosen inhibitor
Specific Aim (3) Synergy with anti-retrovirals: To determine if lead compound(s)
work in synergy with anti-retrovirals with different modes of action,
experiments will be set up with lead compounds in combination with
individual or multiple existing anti-retrovirals.
R5 and X4 viruses will be used
Cytotoxicity of the drug
combinations will be determined
Results will be transferred to Core C for addition to the data
base. Compiled data will used to anticipate drug interactions
in future clinical trials
Specific Aim (4) Loss of susceptibility: HIV will be continuously cultured with
low doses of lead compound. The virus will be periodically tested for
changes in the level of susceptibility to the lead compound.
Compound(s) which make it through the above screening
procedures and are targeted for clinical trials will be tested in long
term culture with a susceptible strain of HIV. Cell cultures will be
maintained with low doses of compound with and without virus.
The virus will be tested in a drug susceptibility assay once a
month using “normal cells” and cells that had been cultured with
the same concentration of compound as virus.
If there is a consistent shift of
the inhibitory concentration with
normal cells the virus will be
sent to Project 1.
If there is a shift with treated cells and
not “normal cells” and there is also a
shift with parent (untreated) virus in
treated cells, the cells with both viruses
will be sent to Project 2 to look for
changes in NK-1R
Specific Aim (5) Efficacy against SIV: SIV strain targeted for use in the
macaque mode will be tested in PBMC against lead compounds for drug
susceptibility.
Viruses will also be isolated from infected macaques and
tested for drug susceptibility in conjunction with parent virus used for initial
infection.
Initial testing results will be forwarded
to Project 3 to facilitate decisions
about drug concentrations to be used
for testing.
Results of these tests will be sent
back to Project 3 via Core C to be
correlated with clinical results.
Specific Aim (6) Sensitivity of patient isolates: To determine the
susceptibility of isolates from patients participating in the clinical trial;
viruses, isolated from individual patients, will be tested in vitro against
the SP antagonist. This will be performed pre and post drug
administration.
Data will be sent to Core C
for correlation with clinical
trial results (Project 4).
Viruses which demonstrate a change in
susceptibility with increasing treatment during the
trial will be evaluated to determine if viral receptor
interactions have changed.
Viruses with changes in receptor
interactions will be sequenced by
Core B