The Toxicologist - Society of Toxicology

Lymphocryptovirus (LCV) in macaques is equivalent to Epstein-Barr virus in humans,
and LCV can cause disease in immunosuppressed macaques that is similar to
that observed in patients. Because LCV can be a serious opportunistic pathogen, it
is useful to monitor LCV in macaques during preclinical development, especially
when immunomodulatory drugs are being used. Here we present the development
of two real time PCR assays to evaluate LCV: a DNA viral load assay and a lytic
gene expression assay for BALF-2. To allow for accurate comparisons between samples,
both assays include endogenous controls for normalization. The specificity of
these assays was confirmed by using peripheral blood mononuclear cells (PBMCs)
from monkeys of known LCV status (+/-) as well as by using a positive control
LCV+ lymphoblastoid cell line (LCL). LCV was detected in the positive control
samples and not detected in samples known to be LCV negative. The virus was also
detected in whole blood, PBMCs and nasopharyngeal swabs of cynomologus
macaques. Overall, as expected there was a higher level of LCV DNA and BALF-2
gene expression detected in nasopharyngeal samples compared to PMBCs and
whole blood. More animals had low or no signal for BALF-2 mRNA when compared
to viral load (DNA). These assays appear to be sensitive for detection of LCV
and therefore can be used to monitor LCV status. We anticipate that such monitoring
will allow us to assess whether LCV is reactivated from latency to lytic phase
replication.
1150 IMMUNOGENICITY TESTING OF INFLIXIMAB
(REMICADE®) AND ADALIMUMAB (HUMERA®) IN
GOTTINGEN MINIPIGS®.
G. J. van Mierlo 1 , A. H. Penninks 1 , J. Wolthoorn 1 , N. H. Cnubben 1 , L.
Aarden 2 and D. Wouters 2 . 1 Toxicology and Applied Pharmacology, TNO-Quality of
Life, Zeist, Netherlands and 2 Sanquin Research, Amsterdam, Netherlands. Sponsor:
R. Woutersen.
As the minipig is currently considered to be a useful alternative species for safety
evaluation of (bio)pharmaceuticals we explored their possibilities for immunogenicity
testing. In this study the potential immunogenicity of two TNFalpha
blockers, Adalimumab and Infliximab both clinically used in chronic inflammation
disorders, is evaluated. These pharmaceuticals differ in biological activity in minipigs
as Adalimumab is active and Infliximab is inactive. Göttingen Minipigs® (4˛
and 4ˇ per group) were treated subcutaneously on days 0, 14, 28, 42 and 56 with
low dose (0.1 mg/kg), mid dose (0.1 mg/kg) or high dose (5 mg/kg) Adalumimab
or high dose (5 mg/kg/day) Infliximab, followed by a 4 wk recovery period. Clinical
signs, injection site reactions, body weight, hematology and the weight and pathology
of several organs were used as criteria for disclosing possible adverse effects.
Blood samples for anti drug antibody (ADA) determinations and pharmacokinetic
(PK) analyses were collected on several days. For PK analysis blood was collected
before and on several time points after the first (d0) and the last dosing (d56). At all
dose levels of Adalimumab ADA’s were present from day 14 onwards. At the lowest
dose the most clear time-related increase in ADA’s was observed in all animals. At
the mid and high dose levels ADA titers were somewhat lower in some animals. PK
analysis after the last injection (d56) showed an immune-mediated clearance of
Adalimumab in 10/12 minipigs. The Infliximab levels in serum were so high that
that they interfered with the ADA analysis (new analysis in progress).
Pharmacokinetic analysis showed that after the last injection of Infliximab (d56) no
evidence of an immune-mediated clearance was observed with still a very long T1/2
(>14 days). The results obtained will be discussed in connection with those observed
in non-human primates and from clinical experience in humans.
1151 IMMUNOGENICITY TESTING OF KINERET® IN
GOTTINGEN MINIPIGS®.
A. H. Penninks 1 , J. Wolthoorn 2 , N. H. Cnubben 1 and G. J. van Mierlo 1 .
1 Toxicology and Applied Pharmacology, TNO-Quality of Life, Zeist, Netherlands and
2 Analytical Research, TNO Quality of Life, Zeist, Netherlands. Sponsor: R.
woutersen.
The aim of this project was to investigate the feasibility of minpigs as alternative
species for immunogenicity testing of biopharmaceuticals. In this study the potential
immunogenicity of the in minipigs biologically active recombinant human IL-
1 receptor antagonist Kineret® was evaluated. Göttingen Minipigs® (3˛ and 3ˇ
per group) were treated daily subcutaneously for 29 consecutive days with vehicle
(water for injection), low dose (0.5 mg/kg/day) or high dose (5 mg/kg/day)
Kineret®. Clinical signs, injection site reactions, body weight and the weight and
pathology of several organs were used as criteria for disclosing possible adverse effects.
Blood samples for anti drug antibody (ADA) determinations and pharmacokinetic
(PK) analysis were collected. For PK analysis blood was collected pre dose
and several timepoints after the first (day 0) and the last (day 28) dosing. Both low
and high dose Kineret®-treated animals showed production of ADA from day 14
onwards. In females ADA production was not dose-related. In males low dose treat-
246 SOT 2011 ANNUAL MEETING
ment resulted on the average in lower ADA levels than high dose treatment. No remarkable
sex-differences were observed in the high dose Kineret® groups for most
PK parameters. In the low dose groups on day 28, the Cmax as well as the AUC for
Kineret® were higher compared to day 0. As the measured T1/2 of Kineret® was
found to be comparable after the first (d0) and the last injection (d28) the ADAs
are considered not neutralizing (determination of neutralizing capacity of the
ADAs in progress). Tmax was found to be approximately 2 hours and the T1/2 between
1.1 and 2.3 hours. The results obtained in respect to the immunogenicity
testing and PK of Kineret® in the minipig were compared with those available in
literature for NHP and humans. In summary it was concluded that comparable results
were obtained in respect to the immunogenicity and PK of Kineret® in
minipigs and non human primates.
1152 ASSESSMENT OF IMMUNE FUNCTION IN CELLS
ISOLATED FROM BRONCHOALVELOAR LAVAGE (BAL)
FLUID, OBTAINED IN-LIFE IN THE DOG.
V. Leighton, A. Head, G. Hale and S. A. Kirk. Covance Laboratories Ltd.,
Harrogate, United Kingdom.
In-life assessment of the immune system is often restricted to blood measurements.
Administration of test item via the inhaled route or when the lung is targeted can
mean any changes in blood are secondary and sampling from BAL fluid is more appropriate.
Sampling of BAL fluid is often performed as a terminal procedure and
sampling during the study requires additional animals. The objective of this study
was to determine the feasibility of repeat in-life BAL in the dog and assessment of
immune function from the isolated cells. BAL sampling was performed on 2 female,
anaesthetised, beagle dogs 1 week apart. Dogs were positioned in left lateral
recumbancy and a bronchoscope introduced to the trachea via an adapter fitted to
the endotracheal tube. The bronchoscope was advanced until the tip was within a
dependent left caudal lobar bronchus. 1.6mL/kg of saline (~37°C) was instilled via
the irrigation channel and the fluid re-aspirated. Immunophenotyping was performed
on resultant cell suspensions by flow cytometry and phagocytic activity assessed
using the Phagotest® kit (Orpegen). Terminal BAL and blood samples were
taken on Day 15 in order to provide comparative data. Phenotyping results showed
the largest BAL population were CD14+/MHC class II+ (40-70%) followed by
CD3+ T cells (10-30%). CD4+ and CD8+ T cells were typically 5-25% and ≥ 5%
of total cells, respectively. Limited numbers of cells isolated from each BAL sample
meant the Phagotest® had to be adapted to determine the optimum level of phagocytosis
possible with the minimum number of cells. Incubation times and cell concentrations
were adjusted on each collection day. Optimal results were achieved at
1000 cells/μL when incubated with e.coli for 30 min (15-20% phagocytic cells).
Phagocytic activity in blood confirmed the assay performed as expected (40-60%
phagocytic cells). In conclusion, in-life sampling of BAL fluid in dogs provides a
method for assessing immune function in the lungs.
1153 TRANSIENT KNOCK DOWN OF CHK1 IN BONE
MARROW HEMATOPOIETIC PROGENITORS IS
LINKED TO BONE MARROW TOXICITY.
B. Jessen and W. Hu. DSRD, Pfizer, San Diego, CA.
Check point kinase 1 (Chk1) is required in both intra-S phase and G2/M checkpoints
in the cell cycle. It is a protein kinase that plays critical roles in maintenance
of genomic stability and transducing DNA damage response. Chk1 is highly expressed
in CD4+ and CD8+ T-cells and hematopoietic stem cells. Chk1 deficiency
has been shown to block T-cell differentiation. Splenomegaly and severe anemia
were observed in a Chk1 haploinsufficiency mouse model. To better understand the
role of Chk1 in bone marrow hematopoiesis, we conducted transient Chk1 gene silencing
in human bone marrow progenitor cells using Chk1 sequence-specific
siRNA and electroporation. Human bone marrow mononuclear cells and CD34+
stem cells were cultured in HPGM media, and induced to undergo differentiation
and proliferation in the presence of appropriate amount of hematopoietic cytokines.
The electroporation procedure with a negative control siRNA was shown
to have minimal impact on cell viability as compared to a non-electroporated control.
At 48hr post electroporation, more than 70% knock down of Chk1 was confirmed
at the mRNA level using real-time RT-PCR and at the protein level using
immunoblotting. On day 6, hematopoietic progenitor cells electroporated with
Chk1 specific siRNA resulted in more than 60% reduction in cell count as compared
to the scrambled control. The inhibition of hematopoietic progenitor differentiation
and proliferation by Chk1 knock down was confirmed using a colony
unit forming assay, where reduced number of erythroid and granulocyte colonies
was observed with Chk1 siRNA treatment. We established in this study an efficient
method to knock down a gene of interest in hard-to-transfect hematopoietic stem
cells and studied the association with bone marrow toxicity. Furthermore, our results
support a direct role of Chk1 in maintaining normal hematopoiesis in the