Nature Medicine
6, 879 - 885 (2000)
doi:10.1038/78638
A controlled trial of intratumoral ONYX-015, a selectively-replicating
adenovirus, in combination with cisplatin and 5-fluorouracil in patients with
recurrent head and neck cancerFadlo R. Khuri1, John Nemunaitis3, Ian Ganly4, James Arseneau3, Ian F. Tannock7, Larry Romel8, Martin Gore5, Janet Ironside6, R.H. MacDougall6, Carla Heise8, Britta Randlev8, Ann M. Gillenwater2, Patricia Bruso2, Stanley B. Kaye4, Waun Ki Hong1
& David H. Kirn91 The University of Texas M. D. Anderson Cancer Center,
Division of Cancer Medicine Houston, Texas 2 The University of Texas M. D. Anderson Cancer Center,
Division of Surgery, Houston, Texas; 3 U.S. Oncology, Dallas, Texas,
4 Beatson Oncology Institute, University of Glasgow,
Glasgow Scotland 5 Royal Marsden Hospital, London,
England, 6 Western General Hospital, Edinburgh,
Scotland; 7 Princess Margaret Hospital, Toronto,
Ontario; 8 ONYX Pharmaceutical, Richmond,
California; 9 Imperial Cancer Research Fund, London,
England
Correspondence should be addressed to Fadlo R. Khuri fkhuri@mdanderson.orgONYX-015 is an adenovirus with the E1B 55-kDa gene deleted, engineered
to selectively replicate in and lyse p53-deficient cancer cells while sparing
normal cells. Although ONYX-015 and chemotherapy have demonstrated anti-tumoral
activity in patients with recurrent head and neck cancer, disease recurs rapidly
with either therapy alone. We undertook a phase II trial of a combination
of intratumoral ONYX-015 injection with cisplatin and 5-fluorouracil in patients
with recurrent squamous cell cancer of the head and neck. There were substantial
objective responses, including a high proportion of complete responses. By
6 months, none of the responding tumors had progressed, whereas all non-injected
tumors treated with chemotherapy alone had progressed. The toxic effects that
occurred were acceptable. Tumor biopsies obtained after treatment showed tumor-selective
viral replication and necrosis induction.Squamous cell carcinoma of the head and neck afflicts an estimated 500,000
patients annually worldwide1,
2. In the United States, the annual
incidence is estimated at 40,300 cases, with 11,700 associated deaths3. Head and neck tumors are known to have p53 mutations in 45−70%
of cases; both alcohol and tobacco use are associated with these mutations4,
5. In tumors with a normal p53 gene sequence, loss of p5
3 function can occur through p53 protein inhibition and/or degradation6,
7. First-line therapy for localized disease is typically surgery
and/or radiotherapy1,
2. Tumors recur in approximately one-third
of patients after first-line therapy, usually in the region of the original
primary tumor or the neck. Severe morbidity due to pain, oropharyngeal and/or
laryngeal obstruction, and resultant difficulties in swallowing and speech
are not uncommon1,
2,
8,
9. Cancer that has recurred, metastasized
or both is considered incurable. Palliative surgery is difficult and disfiguring,
and further radiation therapy is rarely beneficial for more than a few months.
Several chemotherapeutic agents have been used in recurrent squamous cell
carcinoma of the head and neck. Combination chemotherapy regimens have typically
been shown to induce responses in 30−40% of patients, and the time to
tumor progression is often short10,
11,
12,
13. The median survival
time for patients with disease who are treated with chemotherapy is approximately
6 months10,
11,
12,
13,
14,
15,
16, and treatment with chemotherapy
does not substantially improve survival rates16,
17,
18. Patients
with recurrent disease urgently need an effective, local−regional therapy.
ONYX-015 is an adenovirus with the E1B 55-kDa gene deleted, which replicates
and causes cytopathogenicity in certain cancer cells19,
20.
Although pre-clinical in vitro results have varied21,
clinical data obtained with ONYX-015 have been definitive. Selective intratumoral
replication and tumor-selective tissue destruction has been documented in
phase I and II clinical trials of ONYX-015 in patients with recurrent, refractory
squamous cell carcinoma of the head and neck22,
23,
24. However,
durable responses and clinical benefit were seen in less than 15% of these
end-stage patients. As predicted, p53 mutant tumors underwent necrosis at
a higher rate than did tumors with a wild-type gene sequence (58% and 0%,
respectively)23. Both in vitro and nude mouse-human tumor
xenograft model studies have shown additive or potentially synergistic efficacy
of ONYX-015 in combination with cisplatin-based chemotherapy compared with
that of either ONYX-015 or chemotherapy alone19. ONYX-015 was
able to enhance the efficacy of cisplatin both in p53-deficient and p53-functional
tumor cells, in contrast to its activity as a single agent. Sensitization
of p53-functional tumor cells may involve expression of the adenvirus E1A
gene product, which is a potent chemosensitizer25,
26,
27, induction
of high levels of p53 protein28 or both29.
We have undertaken a phase II clinical trial of intratumoral ONYX-015 in
combination with standard intravenous cisplatin and 5-fluorouracil (5-FU)
chemotherapy in patients with recurrent squamous cell carcinoma of the head
and neck. To assess the contribution of ONYX-015 injection to the anti-tumoral
efficacy, we allowed patients with more than one tumor mass to enroll in the
trial; the largest and/or most symptomatic mass was injected with ONYX-015,
but other masses were not. This study design allowed an internally controlled
comparison of the efficacy of chemotherapy alone (in non-injected tumors)
and the combination regimen with ONYX-015 (in injected tumors).
Results
Thirty-seven patients were enrolled in this study (Table
1, patient characteristics). We were able to asses toxic effects in
all patients, and in 30 patients, we could evaluate disease response to treatment.
For seven patients, two cycles of treatment could not be administered and/or
no follow-up tumor measurements could be obtained. The reasons for this included
tumor progression at non-injected sites before response determination of the
injected tumor (n = 4), withdrawal from study because
of bacterial aspiration pneumonia (n = 1), chemotherapy-induced
mucositis (n = 1) and refusal of further treatment
after 1 day (n = 1). All patients were included in
the 'intent-to-treat' analysis. Eighteen of twenty-nine patients
(62%) had neutralizing antibodies against ONYX-015 at baseline (one patient
did not have a baseline serum sample obtained). p53 gene status could
be ascertained in 20 patients; tumor tissue was insufficient for accurate
sequence determination in the other 10 patients. Seven (35%) tumors had mutant
sequences, and thirteen (65%) had wild-type gene sequences.
 | | |
Overall anti-tumoral efficacy
Treatment caused tumors to shrink in 25 of the 30 cases evaluated (Fig. 1b). We documented objective responses (decrease
of 50% or more) of injected tumors in 63% of patients who could be evaluated
(19 of 30). There were 8 (27%) complete and 11 (36%) partial responses. Based
on an 'intent-to-treat' analysis of all 37 patients, the objective
response rate of injected tumors was 53% (19 of 37). Of the 19 patients with
a disease response, 15 had their disease response confirmed 4 weeks or more
after initial observation. Four patients left the study with ongoing disease
response before the 4-week confirmatory measurements, because of tumor progression
at non-injected sites (n = 3) and for surgical resection
of the injected tumor (made possible after the tumor response;
n = 1).
| | |
There was no correlation between response and baseline tumor size, baseline
neutralizing antibody titer, p53 gene status or prior treatment. Patients
with negative baseline neutralizing antibody titers had a response rate of
55% (6 of 11; 4 complete), compared with 67% (12 of 18; 6 complete) for those
with positive titers. p53 mutant tumors had a 71% (5 of 7) response rate,
compared with 69% (9 of 13) for wild-type gene sequence tumors (the p53 gene
sequence could not be determined in 10 tumors). Tumor size did not correlate
with percent of tumor regression (correlation coefficient (R
2), 0.15), and tumors as large as 10 cm in diameter regressed completely.
The length of time to tumor progression for the injected tumors was also
improved (Fig. 2). Based on Kaplan-Meier estimation,
only 17% of injected tumors had progressed 6 months after treatment initiation
(median length of follow-up care was approximately 5 months). None of the
19 tumors with an objective response has progressed so far (again, median
length of follow-up care has been approximately 5 months). The median survival
time for patients on study was 10.5 months. The 12-month survival rate based
on Kaplan-Meier estimation is 32%. Eleven patients had control (non-injected)
tumor masses present during treatment. Despite being generally larger, 9 of
the 11 injected tumors responded, in contrast to 3 of the 11 non-injected
tumors (Fig. 1b). In six patients, the injected
tumor responded and the non-injected tumor did not respond; no patient had
a response in the non-injected tumor without a response in the injected tumor
(P = 0.014, McNemar's test). The time to tumor progression
was also better for injected tumors than for non-injected tumors (
Fig. 2b; P = 0.006, log rank test).
Four patients whose control tumors were refractory to chemotherapy were allowed
to enroll in a 'compassionate use' trial for identical treatment,
using intratumoral ONYX-015 in combination with the same intravenous chemotherapy
regimen. Despite documented resistance to the chemotherapy alone, the ONYX-015
combination therapy resulted in one complete and one partial response.
| | |
Adverse events
Patients reported grade 3 (severe) and grade 4 (life-threatening) adverse
events (Table 2). Fifty-three percent of patients
reported injection site pain; most cases were grade 1 or 2 and lasted 24 hours
or less. A single patient discontinued treatment because of injection site
pain. A substantial minority of patients reported some flu-like symptoms,
including fever (34%), asthenia (47%) and/or chills (24%). Flu-like symptoms
were less frequent here than with an identical treatment regimen given without
chemotherapy in an earlier study23, including fevers (34% compared
with 73%) and chills (24% compared with 50%); concomitant medications, including
glucocorticoids, were equivalent in the two studies. No patients discontinued
therapy on the basis of flu-like symptoms. There was no substantial (grade
3/4) hepatic dysfunction. The most common adverse events (20% or more) whose
relationship to ONYX-015 was categorized as possible, probable, definite or
unable to be determined were injection site pain (45%) and mucous membrane
disorder (21%). Of these two, 29% were grade 3 and 11% were grade 4. No treatment-related
deaths occurred. Grade 3 or 4 events whose attribution to ONYX-015 could not
be ruled out included injection site pain (16%), mucous membrane disorder
(8%), syncope (5%), kidney failure (5%), facial edema (5%) and anorexia (3%).
| | |
Humoral immune responses
Neutralizing antibody titers were positive in 56% of patients before treatment,
and titers increased in all patients after the first treatment cycle of ONYX-015.
Median baseline antibody titer increased from 265 (range, 0−81,920)
to a median of 10,412 (range, 2,366−40,945) at the end of cycle 1 (day
22), and 12,279 (range, 621−81,920) at the end of cycle 2 (day 44).
Titers continued to rise after a second cycle of treatment in most cases.
There was no correlation between neutralizing antibody titers before or after
treatment and efficacy of treatment. Flu-like symptoms (fever and chills)
during the first treatment cycle were less common in patients with positive
neutralizing antibody titers (30%) than in those who had negative titers (63%)
(P = 0.15).
Tissue histology after treatment
We obtained core needle biopsy samples from ten injected tumors during
the first treatment cycle. We were able to use samples from seven tumors for
histology studies; three samples contained necrotic tumor tissue only. We
documented ONYX-015 replication within tumor tissue on day 5 in three of five
tumors and on day 15 in one of two tumors after treatment initiation (Fig. 3). Given the heterogeneity of these tumors and the narrow
width of the biopsy specimens, a negative result for replication does not
rule out the possibility of replication within another section of the tumor.
Substantial necrosis was present adjacent to areas of viral replication, particularly
in the specimen obtained on day 15. All biopsy samples contained a mixture
of normal and tumor tissue; there was no ONYX-015 replication or associated
necrosis in normal tissues within any of the biopsy specimens (
Fig. 3).
Discussion
Combinations of cytotoxic agents in combination with cytolytic viral therapies
have been evaluated30; however, we used a combination of a replication-competent
viral agent and chemotherapy in patients with cancer. The combination treatment
was well tolerated by patients; chemotherapy toxicity was not demonstrably
altered by concomitant viral therapy, and ONYX-015-related flu-like symptoms
were actually less severe than in prior studies with ONYX-015 as 'monotherapy'.
The overall injected-tumor response rate, complete response rate and time
to tumor progression data here were better than the published data from multi-center,
randomized trials of cisplatin and 5-FU (refs. 10, 11, 12, 13, 14, 15, 16). The patient population here was similar to that of
the previous phase II and III trials, and the percent of patients whose disease
responses could be evaluated was similar. However, the tumors described could
all be injected, which might have led potentially to selection bias. We therefore
designed the trial to include internal control tumors whenever possible. To
bias the results against the injection group, we injected the largest or most-symptomatic
tumor with ONYX-015. Despite this intentional bias against the ONYX-015 injection
approach, the injected tumors still achieved objective responses at a substantially
higher rate than that of non-injected tumors within the same patients. In
addition, two of four chemotherapy-refractory tumors responded to subsequent
therapy with ONYX-015 plus the same chemotherapy they had been resistant to.
These results demonstrate tumor-selective augmentation of chemotherapy efficacy
by ONYX-015.
Thus, ONYX-015 combined with cisplatin and 5-FU was active as a method
of local control in most patients. Whether this enhanced local control will
be translated into a survival advantage remains to be confirmed by phase III
trials, but a median survival time of approximately 11 months is encouraging.
The ultimate cause of death in most of these patients was progression at non-injected
sites. If all tumors are injected in future trials, the clinical benefit to
and length of survival of patients may be improved further. Thus, a compelling
hypothesis would be that local control of all disease in the head and neck
region by injection of all clinically detectable sites could improve overall
survival rates. Microscopic sites of disease that are not initially injected
can progress in some patients and become clinically evident over time, as
seen here. Anecdotal data from the 'compassionate use' trial has
shown that patient re-treatment in previously non-injected sites can lead
to objective responses in these newly evident tumors. The clinical benefit
of such re-treatment must be determined in future trials. We have undertaken
a phase III trial of this combination for patients with recurrent head and
neck cancer in whom all clinically evident disease can be injected; the 'control
arm' of the trial will be cisplatin and 5-FU alone. In addition to further
defining the involvement of ONYX-015 adenovirus in the treatment of squamous
cell carcinoma of the head and neck, the function of chemotherapy may be reestablished
as enhancing local disease control, after several decades of disappointing
results.
We documented selective replication within tumor tissue; despite concomitant
treatment with a DNA-damaging agent (cisplatin) and a nucleoside analog (5-FU)
at therapeutic doses. Baseline neutralizing antibody titers did not correlate
with efficacy. In addition, re-treatment resulted in tumor responses despite
as many as five cycles of prior viral therapy and very high neutralizing antibody
titers. Therefore, these data indicate that neutralizing antibodies do not
prevent the efficacy of the intratumoral injection. This finding may result
from inefficient penetration of these solid tumors by neutralizing antibodies31,
32. ONYX-015 administered by a different route (such as intravenously)
may be more sensitive to neutralizing antibody effects. Clinical trials to
answer this are underway.
The ability of tumor cells to develop chemotherapy resistance is a principal
limitation of standard anticancer drugs. As viral therapy and chemotherapy
act by such different mechanisms, it is not unexpected that cross-resistance
mechanisms have not been identified so far. In addition, data from both pre-clinical
models and clinical trials indicate that ONYX-015 may be able to sensitize
infected and uninfected cells to killing by chemotherapy. E1A gene expression
is a potent chemosensitizer25,
26; this effect is independent
of p53 in some models27. As ONYX-015 expresses E1A in both p53-deficient
and p53-functional cancer cells, this mechanism may account for the chemosensitization
of both tumor types in vitro. E1A expression does not chemosensitize
normal, non-transformed cells24. Chemosensitization of uninfected
tumor cells would presumably require a soluble mediator. Adenovirus-induced
cytokines such as tumor necrosis factor can act as potent chemosensitizers33,
34,
35,
36. However, systemic administration of tumor necrosis
factor can cause severe toxic effects in normal tissues37,
38,
39.
Tumor-specific expression of tumor necrosis factor, however, may augment safety
and anti-tumoral effects, and pre-clinical studies are underway to explore
this hypothesis. Elucidation of the mechanisms involved in this chemosensitization
may allow enhancement of this effect in future trials.
Future clinical trials may explore combinations of ONYX-015 by different
routes of administration with other chemotherapy regimens40,
radiation therapy, surgery and even other biological agents. The efficacy
of selectively replicating viruses can also be increased by the expression
of therapeutic transgenes from the virus itself41,
42. It is
likely that such combination approaches will be used to reduce tumor burden
and help restore quality of life in patients with head and neck cancer and
other solid tumors.
Methods:
Enrollment criteria.
All patients had histologically
confirmed squamous cell carcinoma of the head and neck (excluding nasopharyngeal)
that had recurred or relapsed after surgery, radiotherapy or both for the
primary tumor. Tumors could not be surgically curable and could not have received
any chemotherapy subsequent to recurrence; patients with prior 'neoadjuvant'
or adjuvant chemotherapy for their original tumor were not excluded unless
chemotherapy resistance had been documented (based on tumor progression 4
weeks or less after chemotherapy completion). The tumor mass to be treated
with ONYX-015 had to be adequately injectable (described below) and measurable
(radiographically or by physical examination). Patients had to be 18 years
of age or older and were required to have a Karnofsky performance status of
70 or more. Normal hematologic and renal function were also required. The
p53 gene status and p53 immunohistochemical staining were determined for tumors,
but were not used as enrollment criteria. Tumors were deemed 'assessable
for efficacy' if they were given more than one cycle of treatment and
had follow-up measurements on day 45 of the study or later. All patients signed
a consent form approved by the institutional review board committee before
enrollment.
Baseline assessment.
The data obtained in the baseline
assessments made before treatment were not used as enrollment criteria. All
patients had a biopsy sample sent for p53 gene sequencing (described below).
Baseline blood tests included complete blood counts; CD3, CD4 and CD8 lymphocyte
counts; levels of electrolytes, blood urea nitrogen and creatinine; and liver
function tests. In addition, baseline neutralizing antibody titers against
ONYX-015 were determined, because most adults have neutralizing antibodies
against the adenovirus type 5 coat proteins that are present on ONYX-015.
In addition, delayed hypersensitivity skin testing was done and plain chest
radiographs were obtained.
ONYX-015 handling and processing.
ONYX-015 (dl1520)
is a chimeric human group C adenovirus (Ad2 and Ad5) that does not express
the 55-kDa product of the E1B gene. It contains a deletion between nucleotides
2,496 and 3,323 in the E1B region encoding the 55-kDa protein. In addition,
a transition of C to T at position 2,022 in E1B generates a stop codon at
the third codon position of the protein. These alterations eliminate expression
of the E1B 55-kDa gene in cells infected with ONYX-015. ONYX-015 was grown
and 'titered' on the human embryonic kidney cell line HEK293 as
described20. ONYX-015 is provided as a sterile viral solution
in Tris buffer (10 mM Tris, pH 7.4, 1 mM MgCl2, 150 mM CaCl and
10% glycerol), at -20 °C in single-use, plastic screw-cap vials.
Each vial contains 0.5 ml virus solution at a specified viral titer. Vials
of virus solution were warmed and diluted to the appropriate titer for dosing.
The virus-containing solution was then further diluted to a final volume equivalent
to 30% of the estimated tumor volume to be injected. Tumor volume was estimated
by taking the product of the maximum tumor diameter, its perpendicular and
its estimated depth, and dividing by two. Vials of ONYX-015 were opened and
diluted in biological safety cabinets at the patient treatment area immediately
before injection. All waste items were disposed of in biohazard containers
and autoclaved or incinerated.
Treatment regimen.
To ensure uniform dosing to the
injected tumor in each patient and to allow non-injected tumors to serve as
internal controls (if present), a single tumor was identified for ONYX-015
injection in each patient. If more than one tumor that could be injected was
present, the most symptomatic and/or largest tumor mass was injected with
ONYX-015. The tumor was injected with 1  1010 plaque-forming
units per day for 5 consecutive days (Fig. 1a,
injection template). Most of the viral dose was administered at the tumor
periphery and at the interface between normal tissue and tumor; prior studies
have indicated improved efficacy with this administration approach43.
Attempts were made to distribute the virus uniformly along the needle tracks
by gradually depressing the syringe plunger during withdrawal of the needle.
Most of the viral dose was administered at the tumor periphery and at the
interface between normal tissue and tumor43. This approach was
used each day from puncture sites that were equally spaced and encompassed
the entire tumor mass. Chemotherapy was initiated on the same day as the first
ONYX-015 injection as follows: cisplatin (80 mg/m2 intravenously
over 4 h) on day 1 only and 5-FU (800−1,000 mg/m2 continuous
infusion per day) for 5 consecutive days. A dose of 800 mg/m2
5-FU was used if patients had received prior radiotherapy. Treatment cycles
were repeated every 3 weeks until the injected tumor grew or until the patient
experienced intolerable toxic effects; if further chemotherapy was not possible,
ONYX-015 injections could be continued as described above.
Tumor assessments.
Tumor masses were measured serially
by either physical examination or radiographic scanning (computerized tomography
or magnetic resonance imaging), whichever was deemed most accurate for the
measurement of the specific tumor mass. In general, very superficial lesions
were measured by physical examination, and deeper tumors were best measured
by radiographic scanning. Tumors were measured either every 3 weeks (by physical
examination) or every 6 weeks (by computerized tomography or magnetic resonance
imaging scans) while patients were on the active study treatment; after treatment
completion, tumors were assessed every 8 weeks (or sooner if signs/symptoms
of progression became evident). Responses were determined for individual tumors
as follows: Complete response, complete disappearance of measurable tumor;
partial response, decrease in cross-sectional tumor area of 50−100%;
stable disease, decrease less than 50% decrease and increase in tumor area
less than 25%; progressive disease, increase in tumor area versus baseline
area of 25% or more. The time to tumor progression was determined separately
for the ONYX-015 injected site and at non-injected tumor sites, if present.
Progression of non-injected tumors resulted in patients who stopped the study
treatment in several cases, and further follow-up of the injected (responding)
tumor was therefore not possible in all cases. As a result, repeat documentation
of responses 4 weeks later was not required. Response duration was instead
assessed by determining the time to progression of the injected tumors by
Kaplan-Meier analysis; responding injected tumors were 'censored'
for further follow-up once non-injected tumor progression resulted in patients
leaving the study and receiving other therapy. An observation is "censored"
if no event had occured by the end of the observation period of if an intervening
event other than the one of interest occurred. Thus, censored observations
contribute at-risk time, but no events to the estimate of risk.
Internal controls:
ONYX-015-injected and uninjected
tumors. A subset of patients in the study (n = 11)
had measurable secondary (non-injected) tumors identified at baseline. These
patients' tumors were analyzed by comparison of the response rate and time
to tumor progression for the ONYX-015-injected and non-injected tumors (internal
controls). The efficacy of ONYX-015 in combination with chemotherapy was compared
with chemotherapy alone in the same patients. There was no statistically significant
difference in median tumor volumes of the treated and untreated lesion (9.9
cm3 and 3.2 cm3; P = 0.3
), but the larger lesion was selected for injection with ONYX-015 (
P = 0.3). Tumor locations and the frequency of prior irradiation
to the tumor site were similar between the two tumor groups.
Additional follow-up after treatment initiation.
Measurements
of neutralizing antibody titers were repeated every 4 weeks. Repeat tumor
biopsies between days 5 and 22 of each treatment cycle were optional based
on patient consent; these biopsies were mainly obtained after they were judged
to be safe based on a lack of local toxicity with the treatment in the first
patients on study. Biopsies were analyzed for necrosis, inflammatory cell
infiltration and for viral replication by in situ hybridization. Routine
blood testing was repeated every three weeks, including complete blood count,
levels of electrolytes, blood urea nitrogen and creatinine, and liver function
tests.
p53 gene sequencing.
Tumor biopsies for p53 sequencing
were obtained from the recurrent tumor mass that was to be injected before
treatment. Exons 5−9 were sequenced completely during the first two-thirds
of the trial. Approximately 90% of all p53 gene mutations in head and neck
cancers lie within these exons4,
5. 'Gene-chip'
analysis of exons 2−11 was used during the final one-third of the trial.
All wild-type gene sequences obtained with the 'gene chip' required
confirmation by direct sequencing to be considered valid.
In situ hybridization for adenoviral DNA.
Biopsy
samples were used for in situ hybridization adenoviral DNA to determine
the extent of replication of ONYX-015 in both tumor and adjacent normal tissues
as described20,
30. Samples were formalin-fixed, paraffin-embedded
tissue, cut into sections 5  m in thickness. Slides were deparaffinized
in xylene, hydrated with ethanol, digested with proteinase K and 'post-fixed'
in 4% paraformaldehyde. Samples were hybridized overnight at 37 °C with
0.5 g/ml biotinylated adenovirus DNA probe (Enzo Diagnostics, Farmingdale,
New York). After three successive washes in 1X SSC at 55 °C, an alkaline-phosphatase-conjugated
against biotin (Vector Laboratories, Burlingame, California) was applied.
Nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) was
used as the chromagen, and slides were counterstained with nuclear fast red.
Determination of neutralizing antibody titers.
Tumor
and control samples were incubated at 55 °C for 30 min to inactivate complement.
Clinical plasma samples previously determined to produce high, mid-range and
negative titers were designated as plasma controls. Test samples were serially
diluted and mixed with adenovirus stock at a titer pre-determined to produce
15−20 plaques per well in a 12-well dish of JH293 cells. Tumor and control
samples were incubated with virus for 1 h at room temperature and were then
applied to JH293 cells. After 2 h of incubation at 37 °C in an atmosphere
of 5% CO2, the plasma−virus mix was removed, and 2 ml 1.5%
agarose in DMEM was added to each well. The plaque-forming units per well
were counted on plates on day 7 after inoculation. The titer of neutralizing
antibody for each sample was reported as the dilution of plasma that reduced
the number of plaques to 60% of the number of plaques in the virus control
without antibody.
Received 10 March 2000; Accepted 23 June 2000
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Acknowledgments
We thank the following individuals for their assistance on this clinical
trial and on this manuscript: P. Coldiron, D. Davies, O. Diri, J. Kuhn, S.
Landers, T. McCarty, V. Papadimitrakopoulou, M. Posner, M. Propst, P. Roo,
L. Siu, J. Starr, K. Sultan, S. Toney and P. Trown.
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