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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 467-474
GENE THERAPY
Long-term in vivo survival of receptor-modified syngeneic T cells
in patients with human immunodeficiency virus infection
Robert E. Walker,
Christine M. Bechtel,
Ven Natarajan,
Michael Baseler,
Kristen M. Hege,
Julia A. Metcalf,
Randy Stevens,
Allison Hazen,
R. Michael Blaese,
Clara C. Chen,
Susan F. Leitman,
Jolie Palensky,
Janet Wittes,
Richard T. Davey Jr,
Judith Falloon,
Michael A. Polis,
Joseph A. Kovacs,
David F. Broad,
Bruce L. Levine,
Margo R. Roberts,
Henry Masur, and
H. Clifford Lane
From the Clinical and Molecular Retrovirology Section, Laboratory of
Immunoregulation, National Institute of Allergy and Infectious
Diseases, Clinical Gene Therapy Branch, National Human Genome Research
Institute, and the Departments of Nuclear Medicine, Transfusion
Medicine, and Critical Care Medicine, Clinical Center, National
Institutes of Health, Bethesda, MD; SAIC/Frederick, Frederick Cancer
Research and Development Center, Frederick, MD; Cell Genesys, Inc,
Foster City, CA; Statistics Collaborative, Washington, DC; and the
Leonard and Madlyn Abramson Family Cancer Research Institute at the
University of Pennsylvania Cancer Center, Philadelphia, PA.
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Abstract |
To study human immunodeficiency virus (HIV)-specific cellular
immunity in vivo, we transferred syngeneic lymphocytes after ex vivo
expansion and transduction with a chimeric receptor gene (CD4/CD3- )
between identical twins discordant for HIV infection. Single and
multiple infusions of 1010 genetically modified
CD8+ T cells resulted in peak fractions in the
circulation of approximately 104 to 105
modified cells/106 mononuclear cells at 24 to 48 hours,
followed by 2- to 3-log declines by 8 weeks. In an effort to provide
longer high-level persistence of the transferred cells and possibly
enhance anti-HIV activity, we administered a second series of infusions
in which both CD4+ and CD8+ T cells were
engineered to express the chimeric receptor and were costimulated ex
vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions
of approximately 103 to 104 modified
cells/106 total CD4+ or CD8+
cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the
presence of modified cells in proportions equivalent to or below those
in the circulation. The cell infusions were well tolerated and were not
associated with substantive immunologic or virologic changes. Thus,
adoptive transfer of genetically modified HIV-antigen-specific T cells
was safe. Sustained survival in the circulation was achieved when
modified CD4+ and CD8+ T cells were infused
together after ex vivo costimulation, indicating the important role
played by antigen-specific CD4+ T cells in providing
"help" to cytotoxic effectors.
(Blood. 2000;96:467-474)
© 2000 by The American Society of Hematology.
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Introduction |
Antiretroviral therapies have radically altered the
course of patients with human immunodeficiency virus (HIV)
infection.1 However, these treatment regimens, commonly
referred to as highly active antiretroviral therapy (HAART), may be
limited by toxic effects, cost, inconvenience, and incomplete viral
suppression leading to drug resistance. Despite often dramatic
virologic and CD4+ T-cell responses after instituting
HAART, development of effective HIV-specific immune responses and
replacement of elements of the immunologic repertoire depleted by HIV
infection do not occur consistently.2,3 For these reasons,
adjunctive approaches to treatment, including immunomodulatory
therapies, are under investigation.
Virus-specific T cells play an important role in host defense against
certain viral infections. HIV-specific T-cell activity is likely a
major component of the host immune response associated with control of
virus replication after acute infection.4,5 Furthermore,
HIV-specific cytotoxic T lymphocytes (CTL) isolated from infected
patients showed specific cytotoxic activity against HIV-infected target
cells, suggesting a possible role for HIV-specific CTL in more advanced
infection.6 In experiments conducted in simian
immunodeficiency virus (SIV)-macaque models of acute and chronic
lentiviral infection, SIV-specific CD8+ T-cell depletion
with an anti-CD8 antibody was associated with acceleration of death
during acute infection and increased plasma viremia in chronically
infected animals; moreover, control of viral replication in the plasma
was associated with the return of SIV-specific CTL.7,8
These observations suggest that enhanced HIV-specific CTL activity may
be of potential therapeutic benefit in reversing or preventing further
immunologic decline in patients with HIV infection.
How best to augment CTL activity in vivo is unknown. In tissue culture,
in response to stimulation with viral peptides, virus-specific CTL
express cytokines such as interferon  and show cytolytic activity
toward virally infected targets. Evidence of in vivo Epstein-Barr virus
(EBV)-specific9 and cytomegalovirus-specific10 antiviral activity and of EBV-specific anti-tumor
activity11 has been found in patients who have undergone
allogeneic bone marrow transplantation and subsequently received
donor-derived virus-specific CTL. These observations suggest that CTL
can be fully functional on their own. Other studies, however, indicated that CD8+ CTL rely on "help" in the form of
antigen-specific CD4+ T cells and interleukin 2 (IL-2) to
clear chronic viral infections.10,12
T cells bearing chimeric antigen-receptor proteins have been developed
in the laboratory in an effort to redirect T-cell antigen specificity
and circumvent major histocompatibility complex (MHC) restriction. An
example of this technology involves the CD4/CD3- chimeric receptor,
which contains the extracellular targeting domain, human CD4
(accounting for HIV specificity), linked to the cytoplasmic signaling
domain, CD3-, enabling T-cell activation on binding of the receptor
regardless of MHC haplotype.13 On binding to the
HIV envelope, CD8+ T cells genetically engineered to
express the CD4/CD3- receptor proliferate and initiate effector
functions such as cytokine secretion and HIV-specific cytolytic
activity.13,14
To explore the role of HIV-specific T-cell immunity in vivo, we
genetically engineered HIV-specific, CD4/CD3--bearing, syngeneic T
cells obtained from healthy adult donor twins and administered these
cells to their HIV-infected identical twins. We sought to assess the
safety and feasibility of these cell transfers, as well as the in vivo
survival, distribution, and activity of the ex vivo-modified cells in
the setting of HIV infection.
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Patients, materials, and methods |
Patients
Sets of twins were determined to be identical on the basis of HLA
and erythrocyte marker phenotypes. HIV infection in the recipient twin
was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western
blotting. Donor twins were confirmed to be negative for HIV by ELISA,
Western blotting, and polymerase chain reaction (PCR) using 3 sets of
primer pairs amplifying regions for gag, env, and long
terminal repeats (LTRs). Other criteria for enrollment included
seronegativity of the donor for EBV, cytomegalovirus, and hepatitis B
and C viruses, unless the recipient was seropositive for the organism.
Written informed consent was obtained from all participants after the
nature and risks of the study were explained. The study protocol and
procedures were reviewed and approved by the National Institute of
Allergy and Infectious Diseases Institutional Review Board, the
National Institutes of Health (NIH) Institutional Biosafety Committee,
and the NIH Recombinant DNA Advisory Committee.
The first 3 twin recipients enrolled received a single infusion of
107 HIV-specific, genetically modified CD8+
CTL. Thereafter, twin recipients were randomly assigned in a 1:1:1:1
ratio to receive ex vivo-stimulated and -expanded, but unmodified,
CD8+ T cells or either 108, 109, or
1010 HIV-specific, genetically modified CD8+
CTL. After completion of the single-infusion phase of the study, patients randomly assigned to the unmodified-cell group received infusions of 1010 unmodified cells every 8 weeks for 1 to 6 infusions (mean, 5.4 infusions; median, 6 infusions) and patients randomly assigned to the modified-cell group received 2 to 6 infusions (mean, 5.3 infusions; median, 6 infusions) of 1010
HIV-specific, modified cells according to the same schedule. A subset
of 8 patients participated in a second randomization to receive either
modified cells alone or modified cells plus IL-2 infusions. IL-2 was
given by continuous intravenous administration as a starting dose of 9 million IU/day for 5 days beginning the day of each cell infusion, every 8 weeks.
After completing 1 year of treatment, patients were offered an
additional 3 infusions of 1010 HIV-specific, genetically modified CD4+ and CD8+ T cells administered
biweekly on an open-label basis. Patients who received IL-2 with
previous cell infusions received IL-2 with the third of the modified
CD4+ and CD8+ infusions.
CD8+ CTL isolation, enrichment, and growth
Lymphocytapheresis in the donor twins was performed with an
automated cell separator (CS-3000; Baxter Healthcare, Deerfield, IL).
Approximately 10 L of whole blood was processed for each procedure to
collect a minimum of 5 × 109 peripheral blood mononuclear cells (PBMC). CD8+ T cells were enriched with
2-staged immunoselection using immunoaffinity columns (CellPro,
Bothell, WA) containing anti-CD8 monoclonal antibodies (anti-Leu2a;
Becton Dickinson, San Jose, CA) and then CD4 depletion of the effluent
on columns containing anti-CD4 monoclonal antibodies (anti-Leu3a;
Becton Dickinson). A minimum of 5 × 107
CD8-enriched cells were recovered and cultured ex vivo in AR medium
(1:1 AIM V [Gibco, Grand Island, NY]:RPMI [Gibco]-8.8% human AB
serum [Gibco]) supplemented with recombinant IL-2 (700 IU/mL; Chiron,
Emeryville, CA). Cells were stimulated with syngeneic irradiated PBMC
and anti-CD3 antibody (OKT3, 10 ng/mL; Ortho Biotech, Raritan, NJ) and
cultured at 37°C. Cells from twins randomly assigned to the control
group were then expanded to approximately 2 × 1010 total cells.
Growth and transduction of HIV-specific CTL
Cells from the donors of patients randomly assigned to receive
HIV-specific, genetically modified cells were processed as follows.
Supernatant containing the rkat4SVGF3e retroviral
vector15 was added to the cells 5 times during 2 days, for
exposure times of 3, 3, 18, 3, and 3 hours, respectively, on those
days. The rkat4SVGF3e vector contains the following
sequences 5' to 3': pBR322 plasmid backbone; Moloney murine
leukemia virus (MMLV) U3, Moloney murine sarcoma virus LTR, and
5' untranslated region; MMLV env splice acceptor;
CD4/CD3- chimeric-receptor coding sequence; MMLV U3 region; MMLV
LTR; and pBR322 plasmid backbone. The vector titer was 105
to 106/mL, and the multiplicity of infection (MOI)
was 0.7 to 1.4 vector particles/cell (minimum cell number,
5 × 107); the mean transduction efficiency was 18%
(range, 7%-38%). After the final exposure to retroviral vector, fresh
AR medium was added to the cells, which then expanded to a target dose
of 108 cells. A final column purification was performed
with selection for CD4+ expression, thereby capturing a
population of CD8 cells enriched for transduction and expressing both
CD8 and CD4 surface markers. A minimum of 106 transduced
cells were cultured in AR medium containing IL-2 and anti-CD3 to yield
a final cell number of 2 × 1010, with a mean time
in culture of 50 days (range, 35-93 days).
Secondary expansion of purified CD8+ cells
Culture-expanded control cells and HIV-specific transduced cells
were divided into 10 aliquots of approximately
1.5 × 109 each and cryopreserved in liquid nitrogen
in AIM V media with 10% dimethyl sulfoxide (DMSO) (Sigma) and 4.5%
human serum albumin (HSA) (Alpha Therapeutics, Los Angeles, CA). Before
freezing, representative samples were obtained for routine testing for
viability, sterility, and mycoplasma contamination; CTL assays;
and flow cytometry. Testing for replication-competent retrovirus (RCR) was also performed on samples of transduced cells.
Approximately 1 to 2 weeks before infusion, 1 aliquot of cells was
thawed and cultured in AR medium with IL-2 and anti-CD3 to reach the
target cell dose (mean number of days in culture, 12; range, 4-28 days). On the day of infusion, the cells were harvested, filtered
through a 170-µm filter, suspended in 400 mL of normal saline
containing 2.5% HSA, and stored at 2°C to 8°C until infusion.
A cell sample was again tested for viability, percentage of CD4 and CD8
coexpression, cytolytic activity, sterility, mycoplasma, RCR
(transduced cells), and endotoxin contamination. When predefined
release criteria were met (including percentage of total cells
expressing both CD4 and CD8 surface markers  70% and cytolytic
activity against gp120-expressing 293 target cells 100 lytic
units/107 cells), the cells were infused by vein into the
recipient over 1 hour, usually within 4 to 6 hours after they were thawed.
Preparation of CD4/CD3--modified CD4+ and
CD8+ T cells
Donor twins underwent a second lymphocytapheresis as described
above. PBMC were separated with Ficoll gradient separation, and
monocytes were removed by adherence to plastic. The enriched T-cell
population was stimulated to proliferate in serum-free AIM V medium
containing recombinant IL-2 (200 IU/mL) and immunomagnetic beads
(Dynal, Oslo, Norway) loaded by tosyl conjugation with equal amounts of
anti-CD3 OKT3 and anti-CD28 (provided by C. June, Jackson Foundation)
at a ratio of 3 beads/cell.16 On day 3, the stimulated cells were removed by using a magnetic bead separator (MaxSep, Baxter
Healthcare). On days 5 to 7 of stimulation, the cells were exposed to
rkat4SVGF3e retroviral vector supernatant for 3 days at
a MOI of 2, then expanded in serum-free medium containing recombinant IL-2 until a total cell number of at least 2 × 1010
was achieved (mean days in culture, 13; range, 12-17 days). Cells were
then suspended in Plasmalyte A (Baxter) containing 10% DMSO, 1%
dextran (Baxter), and 5% HSA and divided into aliquots of
5 × 109 (50 mL) for cryopreservation. At the time
of cryopreservation, a sample of cells was tested for viability,
sterility, mycoplasma contamination, transduction efficiency,
and RCR and subjected to CTL assays and flow cytometry. For infusion, 2 aliquots (total of 1010 cells) were thawed in a 37°C
water bath and immediately administered to the recipient over 10 to 20 minutes.
Preparation and analysis of radiolabeled cells
An aliquot containing 2.5 × 109 expanded
gene-modified or unmodified CD8+ T cells was removed from
the harvested product. The cells were pelleted, washed, resuspended in
20 mL Hanks balanced salt solution, and incubated for 15 minutes at
room temperature with 0.74 MBq of indium
111-oxine/108 cells (Amersham, Arlington Heights, IL) to a
maximum of 0.37 MBq/kg of body weight. Labeled cells were
then pelleted, resuspended in 100 mL normal saline with 2% HSA, and
filtered through a 140-µm filter (Monoject; Sherwood Medical, St
Louis, MO). The radiolabeled cell suspension was then infused
intravenously over 5 to 10 minutes, and this was followed by infusion
of the remaining unlabeled cells. Approximately 2 to 4, 24, and 48 hours after administration of radiolabeled cells, images were obtained
by using either a BIAD (Trionix, Twinsburg, OH) or Genesis (ADAC Labs,
Milpitas, CA) dual-headed camera equipped with medium-energy
collimators with 20% windows centered at the 174- and 247-keV -ray
energies of indium 111. Whole-body and spot images were obtained.
For analysis, geometric mean images were generated from paired anterior
and posterior whole-body scans. Liver, left and right lung, spleen, and
whole-body regions of interest were drawn on these geometric mean
images, and ratios of organ to whole-body findings were obtained at
each time point.
Analysis of the fraction of CD4/CD3--positive cells
CD4/CD3- DNA copy numbers in peripheral blood and lymphoid tissue
were measured by quantitative competitive PCR using DNA from the
e+ cell line as the competitor. The e+ cell
line contains 10 copies of the CD4/CD3- gene per cell with a
102-base-pair (bp) DNA insert. DNA lysates from varying numbers of
e+ cells were added to equal aliquots of DNA from patient
samples (unfractionated PBMC or lymphoid cells, and immunoselected
CD4+ and CD8+ cells) and amplified for 30 cycles by using Taq polymerase and primer pairs located in the U3
region of the 3' LTR and the CD4/CD3- genes. The primer
sequences used were 5'-GGTTCACTCTTCTCAGCCACTGAAG-3' and
5'-TAGCTTGCCAAACCTACAGGTGGG-3'. These yielded amplified
products of 325 bp from the e+ cells and of 223 bp from
PBMC or lymphoid cells. PCR products were labeled with phosphorus
32-deoxycytidine triphosphate during the amplification and were
electrophoresed on a 10% polyacrylamide gel in 90 mmol/L Tris-borate
and 2 mmol/L EDTA (pH 8.4) buffer. Radioactivity in the resulting bands
was converted to photo-stimulated luminescence units (PSL) by using a
phosphor imager (BAS 1000; Fuji Medical Systems, Stamford,
CT). The CD4/CD3- gene copy number in an individual
sample was derived from a linear regression curve relating the ratio of
PSL for the patient sample to the e+ control on the y-axis
to PSL of the e+ control on the x-axis; the value for the
sample was the corresponding x-axis value to a PSL ratio equal to 1.
Immunoselection for CD8+ T cells from whole blood was
performed by first depleting monocytes from the samples with use of
using anti-CD14-coated beads (Dynal) and selecting CD8+ T
cells with anti-CD8-coated beads. Immunoselection for CD4+
T cells from whole blood was performed by first depleting monocytes and
CD8+ T cells from the samples by using anti-CD14-coated
beads and anti-CD8-coated beads and selecting CD4+ T cells
with anti-CD4-coated beads.
Immunologic and virologic monitoring
Enumeration of fractions of CD3+, CD4+, and
CD8+ subpopulations in processed cells and peripheral blood
samples from the patients was performed by standard 3-color flow
cytometry using the following combination of monoclonal antibodies:
anti-CD3-fluorescein isothiocyanate, anti-CD8-phycoerythrin (Becton
Dickinson), and anti-CD4-Cychrome (Pharmingen, San Diego, CA). HIV RNA
in plasma was quantified with a commercially available reverse
transcriptase-PCR assay (Amplicor HIV Monitor; Roche Diagnostics,
Branchburg, NJ) by using previously described methods with
a sensitivity of 50 copies/mL.17
Statistical analysis
The observed data were derived from sequences of measurements done
over time in the patients in the study. The statistical approaches used
to analyze the data account for the correlation of values within a
patient. Statistical analyses focused on data from the series of
repeated infusions of activated CD8+ T cells and the second
series of infusions of activated CD4+ and CD8+
T cells. Measures of CD4/CD3- gene signal, plasma viral load, and
total CD4+ T-cell count were analyzed.
Values below the detection limit for CD4/CD3- gene signal and viral
load were replaced with random numbers from an exponential distribution
ranging from 0 to the detection limit.18 This method helps
preserve the variability of the measure. Values of 0 were replaced with
one tenth of the minimum of the nonzero values when converting to the
log10 scale. Missing values were imputed by the models
described below or excluded, as appropriate. Wilcoxon rank-sum tests
were used to compare study group populations at baseline.
Persistence of signal over time was modeled with a linear mixed
model18-20 of the log10-transformed measure of
CD4/CD3- gene signal. This approach constructs models that recognize
observations belonging to individual patients but do not make
assumptions about the order and length of time between study points. A
simple variance structure with few assumptions was employed. The
model-based least-squares means at each of the study points were
plotted over time to provide a visual representation of the long- and
short-term effects of repeated infusions of cells. Similar methods were
used to model log10 viral load and total CD4+
T-cell counts over time.
Area-under-the-curve (AUC) analyses were performed for CD4/CD3- gene
signal and viral load. The area for each patient was calculated by
using the trapezoidal rule with the observed values in the original
scale for each time point. The AUC represented the total level of
signal or viral load integrated over time. Two-sample t tests
were used to compare the mean AUC for the different groups. The AUC
analyses included patients who had more than 2 infusions in a series.
Another set of linear mixed models, accounting for order and interval
between points, were fit to log10 CD4/CD3- gene signal, log10 HIV RNA, and total CD4+ T-cell count.
These models recognize the set of observations belonging to a patient
as a unit, and include terms for study group, time point, and the
interaction of group and time point. Slopes of change over time were
calculated by using patients' fitted points from these
interval-adjusted linear mixed models. The model t test for the
interaction term tested the difference in slopes between the groups.
All statistical analyses were conducted with SAS, version
6.12.19
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Results |
Dose escalation of CD4/CD3--modified CD8+ T cells
Twenty-seven subjects participated in the initial dose-escalation
phase of the study: 3 subjects received 107 modified cells
in a nonrandomized pilot phase, and 24 subjects were randomly assigned
to 1 of 4 groups and received single infusions of either gene-modified
cells (10,8 10,9 or 1010 total cells) or unmodified cells (1010). When a quantitative
competitive PCR assay was used to measure CD4/CD3- DNA copy number
in PBMC (with a limit of quantitation of 30 copies/million cells),
CD4/CD3- DNA was detected at the limit of quantitation in 1 of 3 recipients of 107 cells and in 4 of 6 recipients of
108 cells. In 3 of these patients, CD4/CD3- DNA was no
longer detected after the first 1 to 3 days, whereas in 2 patients,
CD4/CD3- DNA persisted for 2 and 24 weeks, respectively.
All 12 subjects who received either 109 or 1010
modified cells had CD4/CD3- DNA detected in their PBMC. Peak copy
number usually occurred 24 to 48 hours after infusion and ranged from 1460 to 41 179 copies/million PBMC. A dose-response relation was evident in that median copy numbers for recipients of 109
and 1010 cells were 2973 and 28 245, respectively.
CD4/CD3- DNA persisted in 11 of 12 evaluable subjects, albeit at
much lower copy numbers (median, 54 copies/million PBMC; range,  30-6587) for at least 15 to 40 weeks, when they received additional
infusions of modified cells.
Repeated infusions of activated CD8+ T cells
Thirty-three patients enrolled in the multiple CD8+
T-cell-infusions phase of the study: 25 had previously participated in the dose-escalation phase, and 8 were newly enrolled and randomly assigned. Three patients did not receive cell infusions during this
study period: 2 died from HIV-related complications before the first
scheduled infusion and 1 voluntarily withdrew from the study. Table
1 shows baseline characteristics of the 30 patients who received at least 1 cell infusion (1010
cells/infusion).
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Table 1.
Baseline characteristics of patients with human
immunodeficiency virus (HIV) infection who received modified or
unmodified CD8+ T cells
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Figure 1A shows the persistence of
CD4/CD3- DNA in the circulation of recipients of either
gene-modified cells or gene-modified cells plus IL-2, expressed as
log10 mean copy number estimated from mixed models. A
reproducible pattern was observed in which copy number peaked 24 hours
after infusion at approximately 105/million
CD8+ cells and then fell steadily to approximately 103
copies before the next infusion 56 days later. By the fourth and
fifth infusions, the nadir copy number in the group of patients who
received cells without IL-2 declined even further. Although mean copy
number for the 4 subjects who received modified cells plus IL-2 also showed 2-log declines from peak values in the days after infusion, this
group had a numerically higher mean nadir DNA copy number. Analysis of
the model-based least-squares means, excluding the day 1 peaks for both
groups, yielded slopes of 0.0009 ± 0.0013 and 0.0088 ± 0.0013 log10 copies/day for the IL-2 recipients and
nonrecipients, respectively (P = .0012 for the interaction of
study time and IL-2 use). An AUC analysis showed no difference between
the 2 groups (P = .48 by 2-sample t test).
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| Fig 1.
Results of repeated infusions of receptor-modified
syngeneic T cells.
(A) Persistence of CD4/CD3--modified CD8+ T cells in
the 17 patients who received gene-modified CD8+T cells
without interleukin 2 (IL-2) (closed squares and solid line) and in the
4 patients who received gene-modified CD8+ T cells with
IL-2 (open circles and broken line). Quantitative competitive DNA
polymerase chain reaction (PCR) for the CD4/CD3- gene was performed
on peripheral blood mononuclear cells (PBMC), and results were
expressed as log10 DNA copies per million CD8+
T cells. The closed triangles spaced at 56-day intervals represent the
timing of each cell infusion with and without IL-2. Data
were modeled for individual patients, and a mixed-model analysis was
used to derive the mean of each time point for the 2 groups. SEs are
between 0.40 and 0.52 at each point. (B) Persistence of
CD4/CD3--modified CD4+ T cells (closed squares and
solid line) and CD8+ T cells (open circles and broken line)
in the 14 patients who received modified CD4+ and
CD8+ cells without IL-2. Data were modeled as described for
Figure 1A and results expressed as log10 DNA copies per
million CD4+ or CD8+ cells. The closed
triangles spaced at 14-day intervals represent the timing of the 3 cell
infusions. SEs are between 0.02 and 0.03 at each point. (C) Mean plasma
human immunodeficiency virus (HIV) RNA levels in recipients of modified
CD8+ T-cell infusions (closed squares and solid line,
n = 17) and in recipients of unmodified CD8+ T cells
(open circles and broken line, n = 9). Data were modeled as described
for Figure 1A and results expressed as log10 HIV RNA copies
per milliliter. Closed triangles represent the timing of cell
infusions. P = .29 for the differences between the slopes of
the 2 curves estimated with an interval-adjusted linear mixed model.
(D) Mean CD4+ T lymphocyte counts in recipients of modified
CD8+ T-cell infusions (closed squares and solid line,
n = 17) and in recipients of unmodified CD8+ T cells
(open circles and broken line, n = 9). Data were modeled as described
for Figure 1A and results expressed as cells per
milliliter. Closed triangles represent the timing of cell
infusions. P = .89 for the difference between the slopes of
the 2 curves estimated with an interval-adjusted linear mixed model as
described for Figure 1C.
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We considered that if IL-2 was promoting cell survival, it was likely
doing so by substituting for HIV-specific CD4+ T-cell help.
Therefore, a second series of cell infusions was done, in which
CD4/CD3--modified CD4+ and CD8+ T cells
were given together. Seventeen of the 30 subjects who were given
repeated infusions of CD8+ T cells participated in this
nonrandomized and uncontrolled study; Table
2 shows their baseline characteristics
before they received the modified CD4+ and CD8+
cell infusions. Four of these patients had previously been given CD8+ cells plus IL-2, and in this phase of the study, 3 received IL-2 with the third of 3 CD4+ and CD8+
cell infusions.
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Table 2.
Baseline characteristics of the 17 patients with HIV
infection who received gene-modified CD4+ and
CD8+ T cells
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When CD4/CD3--modified CD4+ and CD8+ T
cells (1010 cells/infusion) were administered at the same
time, a persistence pattern different from that resulting from modified
CD8+ T-cell infusions was observed (Figure 1B). Of note,
these cells were stimulated ex vivo by using costimulation through CD3
and CD28 receptors16 rather than by anti-CD3 and IL-2
stimulation. CD4/CD3- DNA copy number in circulating
CD4+ and, to a lesser extent, in CD8+ T cells
increased slightly in the interval between cell infusions (Table
3). The persistence curves for the 3 patients who received exogenous IL-2 and cells were indistinguishable
from the curves of patients who received cells alone (data not shown).
Gene-containing peripheral blood CD4+ and CD8+
T cells were detected for at least 1 year after the cell infusions, in
fractions ranging from 45 to 44 208 DNA copies/million
cells.
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Table 3.
Survival of CD4/CD3--transduced peripheral blood
mononuclear cells in 14 recipients of gene-modified CD4+
and CD8+ T cells
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In vivo trafficking of cells
To study homing patterns of the infused cells, an aliquot of ex
vivo-expanded CD8+ T cells was labeled with indium
111-oxine before infusion in 4 subjects, and -camera imaging was
performed approximately 2 to 4, 24, and 48 hours after the cells were
given. Figure 2 shows results from 1 representative series of scans. Radiolabeled cells initially
distributed to reticuloendothelial organs (liver and spleen) and the
lungs. By 20 and 43 hours, the lungs had substantial clearing, whereas
the liver and spleen continued to show preferential uptake. At these
later time points, the labeled cells also distributed to the bone
marrow (spine and iliac bones). Peripheral lymph nodes showed no
evidence of preferential uptake of the labeled cells at any of the time
points studied. The distribution pattern of the CD8+ T
cells in this study was similar to that described after autologous PBMC
infusions in healthy volunteers,21 in whom cells were
neither activated nor expanded ex vivo, and in HIV-infected patients
given ex vivo-activated, autologous CD8+
cells.22
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| Fig 2.
Whole-body -camera images from 1 representative
patient who received 2.5 × 109 indium
111-oxine-labeled CD4/CD3--modified CD8+ T
cells in a total of 1010 gene-modified cells at time 0.
The images shown are anterior projection geometric means from 4, 20, and 43 hours after infusion of cells.
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Lymph node or tonsillar biopsies were performed in 5 patients between 3 and 21 weeks after infusion of CD4/CD3--transduced CD8+
T cells to detect the presence or absence or preferential lymphoid distribution of the transduced cells. In all patients studied, CD4/CD3- DNA copy numbers in tissue were either equivalent to or
below simultaneously determined values in PBMC and were usually below
the limit of quantitation (< 30 copies/million cells) for the PCR
assay (Figure 3A-B). We also assessed in
vivo trafficking in 2 patients who received gene-modified
CD4+ and CD8+ T cells. A lymph node biopsy was
performed in 1 patient 2 weeks after cell infusion, and a tonsillar
biopsy was performed 1 day after infusion in the other patient. As was
observed after infusion of CD8+ T cells alone,
CD4/CD3--containing T cells were present in these lymphoid tissue
samples at fractions equal to or below that in simultaneously collected
PBMC (Figure 3C-D).
View larger version (54K):
[in this window]
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| Fig 3.
Comparison of CD4/CD3- DNA copy number obtained from
PBMC and lymphoid tissue by quantitative competitive PCR.
(A) PBMC sample (142 copies/million cells) and (B) lymph node purified
CD8+ cell fraction (below the limits of quantitation)
obtained from 1 patient 10 weeks after an infusion of 109
CD4/CD3--modified CD8+ T cells. Similar data are shown
in panel C for PBMC (> 5000 copies/million cells) and in panel D for
unfractionated mononuclear cells (MC) from tonsillar tissue (567 copies/million cells) for a different patient 2 weeks after an infusion
of 1010 CD4/CD3--modified CD4+ and
CD8+ T cells. The e+ cell line, which contains
10 copies of the CD4/CD3- gene with a 102-base-pair DNA insert per
cell, was used for standardization.
|
|
Virologic and immunologic effects of the cells
As shown in Figure 1C, a small decline in mean HIV RNA levels
occurred in patients who received repeated infusions of either modified
CD8+ cells or unmodified CD8+ cells (slope,
0.0022 log10 HIV RNA copies/day for the
modified-cell group versus 0.0006 copies/day for the
unmodified-cell group; P = .29 by t test for
interaction in the interval-adjusted linear mixed model). Although
(despite randomization) the groups differed in their baseline mean
total CD4+ T-cell counts, the slopes of the 2 CD4+ T-cell curves were nearly the same (slope, 0.28 cells/day for the modified group versus 0.30 cells/day for the
unmodified group; P = .89 by t test for the
interaction in the interval-adjusted linear mixed model; Figure 1D).
The 4 patients who received modified CD8+ cells and IL-2
infusions during this period had similar changes in mean plasma HIV RNA
levels (slope, 0.0007 log10 copies/day) while
showing the expected IL-2-induced rise in mean CD4+ T-cell
count over the year (slope, 1.44 cells/day).23
In the patients who received modified CD4+ and modified
CD8+ cells without IL-2, HIV RNA levels were essentially
unchanged from baseline values at 6 and 10 weeks after the start of
cell infusions (baseline log10 copies/mL, 2.87 ± 0.38;
week 6 log10 copies/mL, 3.03 ± 0.41; and week 10 log10 copies/mL, 2.81 ± 0.39), whereas CD4 counts
increased from a mean of 390 ± 71 at week 0 to 503 ± 77 at week 6 and to 471 ± 74 at
week 10. Three patients received an infusion of exogenous IL-2 at week
4 with the third infusion of modified CD4+ and modified
CD8+ cells. By week 10, CD4 counts had increased in all 3 patients by 166 to 509 cells/µL; in the 2 patients with
detectable plasma viremia (baseline HIV RNA, 2.97 and 3.40 log10 copies/mL, respectively), HIV RNA levels fell 0.46 and 1.64 log10, respectively, by week 10.
Clinical effects and tolerance of the cell infusions
A total of 212 T-cell infusions were given (161 of
CD8+ cells and 51 of CD4+ plus
CD8+ cells). No serious adverse event related to the cell
infusions occurred. Minor side effects, such as fever, chills, and
other events listed in Table 4, accompanied
28% of the infusions (occurrence rate, 30% for CD8+ T
cells and 22% for CD4+ plus CD8+ T cells). No
cell infusion was interrupted because of side effects. Premedication
with acetaminophen, ibuprofen, or meperidine was offered to patients
who previously had fever, chills, or other influenza-like symptoms with
infusion.
Although this study did not have enough power to reliably detect
differences in clinical end points between groups, HIV-related clinical
events and deaths were recorded. No major differences between the
modified-cell and unmodified-cell groups were observed. In the
modified-cell group, 1 case each of non-Hodgkin lymphoma (NHL),
progressive multifocal leukoencephalopathy (PML), and anorectal carcinoma occurred. In the unmodified group, 2 cases of presumptive PML
(resulting in 1 death) and 1 case of NHL (also resulting in death) occurred.
| |
Discussion |
We demonstrated that genetically engineered, HIV-specific, ex
vivo-expanded and -activated CD8+ and CD4+
cells can be administered safely at doses of up to 1010
cells without producing evidence of substantive acute or cumulative toxicity. We did not study cell numbers above 1010 in this
trial, so the maximal tolerated dose was not determined. HIV-specific CD8+ T-cell therapy resulted in long-term persistence of
gene-modified cells in the bloodstream, but the fraction of
gene-modified cells declined with time, even during the course of
repeated cell administrations. This observation raises the possibility
that immune mechanisms leading to shortened cell survival with
reexposure may be a limiting factor.24,25 Providing
CD4+ T-cell help in the form of exogenous IL-2 infusions or
HIV-specific, CD4/CD3--modified CD4+ T cells appeared
to alter the in vivo survival of the modified cells. This finding was
particularly striking when HIV-specific CD4+ T cells were
coadministered with CD8+ T cells after ex vivo
costimulation with anti-CD3 and anti-CD28; in this setting, transduced
CD8+ and CD4+ T-cell fractions increased
transiently and persisted at relatively high levels (0.1%-0.01% of
circulating cells) for many months. We also showed, using indium 111 radiolabeling studies, that ex vivo manipulation, including cell
transduction, did not alter the typical tissue-distribution pattern
observed with nonmanipulated PBMC in healthy volunteers21
or ex vivo-expanded autologous CD8+ T-lymphocyte infusions
in patients with HIV infection.26
We used the least-squares method with linear mixed-effect modeling to
estimate the immunologic and virologic effects of the cell infusions
and to characterize the persistence of gene-modified cells in the
circulation. We also imputed values for CD4/CD3- DNA and HIV RNA
levels below detection limits by substituting random numbers from an
exponential distribution ranging from 0 to the detection limit. These
complicated approaches were important for this study because they
account for interdependence of longitudinal observations within
patients and the heavy-tailed distributions of the variables
being analyzed while not underestimating variance.18 In the
case of HIV RNA, when we repeated the analysis substituting 49 for each
value below 50 copies/mL rather than using exponential replacements,
the least-squares means were slightly higher and the SEs slightly
smaller; the overall difference in the results was small, however, and
did not change our conclusions.
The cells administered in this study did not appear to have an
antiretroviral effect in vivo. Other investigators have reported similar findings when HIV p24 antigenemia or plasma HIV RNA levels were
assayed after treatments with either polyclonal, ex vivo-expanded CD8+ cells,22,27 HIV-specific CTL
lines,28 or HIV-specific CTL clones.29,30 One
patient who received infusions of an autologous Nef-specific CTL clone
and IL-2 had clinical deterioration.29 Several
possibilities could account for the lack of correlation between in
vitro and in vivo cytotoxicity. Suppression of transcription from the
transgene resulting in gene silencing was one
consideration.31 We analyzed a small number of PBMC samples
from our patients and were consistently able to detect CD4/CD3-
messenger RNA, suggesting that this was not a major factor (data not
shown). Apoptosis is another mechanism purported to account for lack of
in vivo killing32-34; however, survival of transduced cells
would be expected to shorten in this instance and this was not observed
in our study, particularly when CD4+ and CD8+ T
cells were coadministered. A survival pattern consistent with immune
elimination24,25 was observed with the CD8+
T-cell infusions alone, and this might have been responsible for the
limited activity of those cells. However, this same survival pattern
was not evident when CD4+ and CD8+ T cells were
given together or when CD8+ T cells were given with IL-2,
raising the possibility that IL-2-induced anergy to the CD4/CD3-
transgene accounted for increased cell survival.35
Optimal survival of genetically engineered HIV-specific CTL was
achieved by culturing the cells with anti-CD3 and anti-CD28 costimulation16 and coadministering the gene-modified
CD4+ and CD8+ T cells. Costimulation was shown
previously to enhance ex vivo expansion of CD4+ T cells, to
provide transient resistance of the cells to HIV infection, and to
prevent antigen-induced apoptosis.16,36 Future studies
investigating the potential therapeutic role of culture-expanded CTL
should incorporate these modifications. The lack of measurable in vivo
antiretroviral activity remains unexplained. Studies in progress will
explore potential antiviral activity by evaluating HIV-specific CTL
therapy in the setting of controlled viremia and measuring HIV load in
compartments and cell populations believed to represent latent
reservoirs of replication-competent HIV.
| |
Acknowledgments |
We thank the patients and their referring physicians for their
willingness to participate in and support this study; the staff of the
NIAID inpatient unit and the outpatient research clinic of the NIAID
and Critical Care Medicine Department; and A. S. Fauci, R. A. Morgan, and C. S. Carter for their helpful discussions and support.
| |
Footnotes |
Submitted November 16, 1999; accepted March 7, 2000.
Funded in part by the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, under contract NO1-C0-56000.
Reprints: H. Clifford Lane, Building 10, Room
11S231, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1894; e-mail: clane{at}niaid.nih.gov.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction