IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY
Incubating AAV2eGFP with puried complement proteins capable
of generating iC3b on the capsid surface (C3b, plus factor H and
factor I) signicantly reduced transduction of Hela C12 cells cultured
in the presence of mCRIg-CHO, but not HeLa C12 cells cultured
in the presence of CHO K1 cells. Vectors opsonized with C3b, or
control vectors not treated with serum complement components
were unaffected by the presence of CRIg-expressing cells. Although
this data strengthens our hypothesis that clearance of AAV vectors
by tissue-resident macrophages is mediated by complement, it also
delineates the different receptors that mediate host innate responses
to AAV, and suggests a delicate balance between inammatory and
non-inammatory host responses to AAV.
548. Overcoming Anti-AAV Neutralizing
Antibodies with Alternate Serotype and
Pharmacological Strategies
Federico Mingozzi,1 Margriet J. Vervoordeldonk,2,3 Yifeng Chen,1,4
Shyrie Edmonson,1,4 Rogier Thurlings,3 Katherine High,1,4 Paul P.
Tak.2,3
1
Center of Cellular and Molecular Therapeutics, The Children’s
Hospital of Philadelphia, Philadelphia, PA; 2Arthrogen BV,
Amsterdam, Netherlands; 3Div of Clinical Immunology and
Rheumatology, AMC/University of Amsterdam, Amsterdam,
Netherlands; 4Howard Hughes Medical Institute, Philadelphia, PA.
Humoral immunity against adeno-associated virus (AAV) vector
is an important barrier to gene transfer, resulting in clearance of
the vector before it enters the target cell. When administering AAV
vectors directly to the blood stream, anti-AAV neutralizing antibody
(NAb) titers as low as 1:5 can completely ablate vector transduction
capability. Another complication comes from the fact that NAb
directed to the AAV capsid are highly prevalent in humans and
cross-recognize alternate capsids by a variable degree depending
on the homology of capsid amino acid sequence. Administration of
AAV vectors by direct intramuscular injection, or to closed body
compartments, like the eye and the brain, seems to be less susceptible
to neutralization. Since little is known about the effect of NAb on
intra-articular vector administration, we tested a cohort of subjects for
anti-AAV NAb using an in vitro assay with an AAV vector expressing
b-galactosidase as reporter gene. In serum, anti-AAV-2 antibodies
(Ab) were the most prevalent, with 9/11 subjects with a titer >1:10,
compared to 3/11 for AAV-5, 7/11 for AAV-6, and 6/11 for AAV-8.
Ab titer in synovial uid, measured with a capture assay, was lower
than in the matched serum samples (average of 1714 vs. 2337 ng/
ml IgG, respectively, p<0.05) indicating a difference in distribution
of Ab to AAV. We next evaluated the impact on Nab to AAV of a B
cell-targeted therapy in rheumatoid arthritis patients who received
an anti-CD20 monoclonal Ab as part of their disease management.
Subjects received two infusions with the anti-CD20 Ab; NAb before
and after each infusion. When anti-AAV-2 NAb were tested, a drop of
neutralizing titer was observed, which was more marked in subjects
with titers <1:100. Using a luciferase-based assay, which showed high
sensitivity also with AAV serotypes poorly transducing in vitro, we
measured anti-AAV-5 NAb. Anti-AAV-5 Ab showed a drop in 6/15
subjects after the rst course of anti-CD20 Ab; in these subjects NAb
titer remained constant after the second course of B cell depletion.
Only in one subject a further drop was noted after the second round of
anti-B cell therapy. These data show that serotypes other than AAV-2
may help escaping Ab neutralization, with AAV serotype 5 showing
the lowest level of neutralization. In conclusion, anti-CD20 antibody
therapy results in a decrease in NAb titer, more efcient at lower anti-
AAV Ab levels. These data suggest that alternate serotypes, together
with pharmacological treatment, may be an effective approach to
overcome humoral immunity to the AAV vector capsid.
S212
549. Switching the Fiber Knob of Oncolytic
Adenoviruses To Avoid Neutralizing Antibodies in
Human Cancer Patients
Mari Raki,1,2 Iulia Diaconu,1,2 Merja Sarkioja,1,2 Sophie
Escutenaire,1,2 Lotta Kangasniemi,3 Anna Kanerva,1 Vincenzo
Cerullo,1,2 Timo Joensuu,4 Sari Pesonen,1,2 Akseli Hemminki.1,2
1
Cancer Gene Therapy Group, Transplantation Laboratory,
Haartman Institute and Finnish Institute of Molecular
Medicine, University of Helsinki, Helsinki, Finland; 2HUSLAB,
Helsinki University Central Hospital, Helsinki, Finland;
3
Oncos Therapeutics Inc., Helsinki, Finland; 4International
Comprehensive Cancer Center Docrates, Helsinki, Finland.
Neutralizing antibodies (NAbs) may impact the utility of oncolytic
adenoviruses for treatment of cancer. Preclinical reports indicate that
NAb response can be partially overcome by modifying the adenoviral
ber knob. In the current study, 24 cancer patients were treated with
two cycles of oncolytic adenoviruses featuring three capsid variants:
unmodied Ad5, Ad5 with RGD motif in the HI-loop (Ad5-RGD)
and serotype chimeric Ad5/3. A different capsid was used for the
second round of treatment. When analyzed just before the second
cycle, a differential in serum NAb titer against the rst versus second
virus was seen in 83% of cases suggesting that changes in the ber
knob can circumvent neutralization in cancer patients. This may
have implications for treatment of human cancer with oncolytic
adenoviruses.
550. Depletion of B Cells by Anti-CD20 Partially
Regulates Anti-FVIII Antibody Production in the
Nonviral Gene Therapy Model
Peiqing Ye,1 Baowei Peng,1 Marilyn Kehry,2 David J. Rawlings,1,3
Carol H. Miao.1,3
1
Seattle Children’s Research Institute, Seattle, WA; 2Biogen Idec
Inc., Cambridge, MA; 3Pediatrics, University of Washington,
Seattle, WA.
Anti-CD20 binds to pan B cells and induces rapid in vivo depletion
of mature human B lymphocytes. It was hypothesized that concurrent
administration of high-dose FVIII and anti-CD20 is benecial to
hemophilia patients with inhibitors. Administration of anti-murine
CD20 into a group of hemopohilia A mice at 250µg each at days 0
& 14 depleted the CD19+ B cells from 60% to 16% at day 3 and this
level further reduced to 4% at day 14 in blood. Signicant reduction
of B lymphocytes was also observed in the spleen and lymph nodes.
The depletion of B cells lasted for sustained periods of time and
gradually returned to normal at ∼8 weeks post anti-CD20 treatment.
We tested if administration of anti-murine CD20 can successfully
prevent the formation of anti-FVIII antibody following gene
transfer in hemophilia A mice. Fifty µg of pBS-HCRHPI-FVIIIA
were injected into 4 groups of hemophilia A mice (n=5/group) using
hydrodynamics-based delivery at day 0 with concomitant intravenous
injection of anti-CD20 (100µg each at days 0,2,4,7 & 10; 250µg each
at days 0 & 14; 500µg at day 0) or IgG control antibody (500mg at
day 0) through the tail vein. In IgG control group, anti-FVIII antibody
appeared quickly at 2 weeks post plasmid injection, increased to
high-titers at 3-4 weeks, and maintained at high-titer levels through
24 weeks of the experimental period while initial high levels of
FVIII activity decreased to low/undetectable levels within 4 weeks.
In three anti-CD20 treatment groups, one mouse from each group
had persistent FVIII activity without detectable inhibitory anti-FVIII
antibodies. The rest of the treated mice had delayed immune responses
however all generated moderate to high titers of inhibitory antibodies
with FVIII activity decreasing to undetectable levels at 6-15 weeks.
A reduction of inhibitory antibody titers was observed following
anti-CD20 treatment; however, the titer rose back when anti-CD20
decreases over time. In these cases, depletion of B cells following gene
Molecular Therapy Volume 18, Supplement 1, May 2010
Copyright © The American Society of Gene & Cell Therapy