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Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 785-793
PLENARY PAPER
Prolonged survival and tissue trafficking following adoptive
transfer of CD4 gene-modified autologous CD4+ and
CD8+ T cells in human immunodeficiency virus-infected
subjects
Ronald T. Mitsuyasu,
Peter A. Anton,
Steven G. Deeks,
David T. Scadden,
Elizabeth Connick,
Matthew T. Downs,
Andreas Bakker,
Margo R. Roberts,
Carl H. June,
Sayeh Jalali,
Andy A. Lin,
Rukmini Pennathur-Das, and
Kristen M. Hege
From the University of California, Los Angeles, CA; San Francisco
General Hospital, San Francisco, CA; Massachusetts General Hospital,
Boston, MA; University of Colorado Health Science Center, Denver, CO;
Statistics Collaborative, Washington DC; Specialty Labs, Los Angeles,
CA; University of Virginia, Charlottesville, VA; University of
Pennsylvania, Philadelphia, PA; and Cell Genesys, Inc, Foster City,
CA.
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Abstract |
We have genetically engineered CD4+ and
CD8+ T cells with human immunodeficiency virus (HIV)
specificity by inserting a gene, CD4 , containing the extracellular
domain of human CD4 (which binds HIV env) linked to the zeta
() chain of the T-cell receptor (which mediates T-cell activation).
Twenty-four HIV-positive subjects received a single infusion of 2 to
3 × 1010 autologous CD4-modified CD4+
and CD8+ T cells administered with (n = 11) or
without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts
greater than 50/µL and viral loads of at least 1000 copies/mL at
entry. T cells were costimulated ex vivo through CD3 and CD28 and
expanded for approximately 2 weeks. CD4 was detected in 1% to 3%
of blood mononuclear cells at 8 weeks and 0.1% at 1 year after
infusion, and survival was not enhanced by IL-2. Trafficking of
gene-modified T cells to bulk rectal tissue and/or isolated lamina
propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year. A greater than 0.5 log mean decrease in
rectal tissue-associated HIV RNA was observed for at least 14 days,
suggesting compartmental antiviral activity of CD4 T cells.
CD4+ counts increased by 73/µL at 8 weeks in the group
receiving IL-2. There was no significant mean change in plasma HIV RNA
or blood proviral DNA in either treatment arm. This sustained,
high-level persistence of gene-modified T cells demonstrates the
feasibility of ex vivo T-cell gene therapy in HIV-infected adults and
suggests the importance of providing HIV-specific T-helper function.
(Blood. 2000;96:785-793)
© 2000 by The American Society of Hematology.
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Introduction |
Much experimental and observational data suggest that
the T-cell immune response plays a major role in containment of human immunodeficiency virus (HIV) during acute and chronic infection. The
emergence of HIV-specific CD8+ cytotoxic T lymphocytes
(CTLs) coincides with the rapid decrease in plasma viremia during acute
infection, and the frequency of HIV env-specific CTLs is
inversely correlated with plasma viral load and the rate of decline in
CD4+ T-cell counts.1,2 Furthermore, a decline
in HIV-specific CD8+ T cells occurs in patients as they
progress to later stages of the disease.3 Most
HIV-1-infected long-term nonprogressors have high circulating levels
of HIV-1-specific CTL precursors,4,5 and increased
HIV-specific CTLs as well as high HIV-specific CD4+ T-cell
proliferative responses have been demonstrated in HIV-exposed seronegative subjects.6,7 The most direct evidence of the role of CD8+ T cells in HIV infection comes from recent
studies of CD8+ T-cell depletion in acute and chronically
infected macaques with simian immunodeficiency virus (SIV). In the
setting of chronic SIV infection, CD8+ T-cell depletion led
to a rapid 1- to 4-log increase in plasma viral load, which returned to
baseline coincident with recovery of CD8+ T cells in the
blood.8 Following acute SIV infection, CD8+
T-cell depletion led to longer persistence of high-level viremia and a
more rapidly progressive disease course compared with nondepleted monkeys.9
Animal and human experiments in chronic viral infection have
demonstrated that antigen-specific CD4+ T-cell responses
are critical for maintenance of CTLs and eradication of viral
infection.10,11 Most studies have shown HIV-specific CD4+ T-cell proliferative responses to be absent or low in
patients with chronic, progressive HIV infection.12,13
Following initiation of combination highly active antiretroviral
therapy (HAART), the recovery of HIV-specific
CD4+ T-helper cell responses has been observed only in
patients treated very early after acute infection.14
Vigorous HIV-specific CD4+ responses have been associated
with control of viremia in the absence of drug therapy and clinical
long-term nonprogression.14 Although recent flow
cytometry-based studies have demonstrated the presence of HIV-specific
CD4+ T cells in the majority of actively infected
individuals, functional enhancement is still likely to be necessary for
optimal defense against HIV.15 In total, these data suggest
that both arms of the host cellular immune response are necessary for
containment of HIV.
Immunotherapy of viral infection with the use of antigen-specific T
cells has been studied in the setting of cytomegalovirus (CMV),
Epstein-Barr virus (EBV), and HIV. Adoptive transfer of allogeneic
CMV-specific10,16 or EBV-specific17-19 T cells
in bone marrow transplant recipients has resulted in recovery of virus-specific CTL activity, reduction of viremia, and effective prophylaxis or treatment of CMV- and EBV-induced disease. Endogenous recovery or adoptive transfer of antigen-specific CD4+ T
cells was required for long-term maintenance of transferred CMV- or
EBV-specific CTLs.19,20 Thus, adoptive immunotherapy with
antigen-specific T cells is likely to require co-infusion of
CD4+ and CD8+ T cells to achieve optimal in
vivo survival and activity.
Adoptive transfer of HIV-specific T cells has potential as
immunotherapy for HIV infection. Strategies to date have focused on ex
vivo-expanded autologous HIV-specific CD8+ monoclonal or
polyclonal T cells.21-24 Rather than isolating and expanding rare T-cell clones with major histocompatibility complex (MHC)-restricted antigenic specificity, a method has been developed to
generate large numbers of HIV-specific primary T cells rapidly using
retroviral-mediated gene transfer to insert an HIV-targeting gene
(CD4).25 CD4 is a genetically engineered,
MHC-unrestricted, chimeric immune receptor composed of the zeta ()
subunit of the CD3 T-cell receptor (the cytoplasmic domain involved in
signal transduction) fused to the transmembrane and extracellular
domains of human CD4 (which targets HIV env expressed on the
surface of infected cells). The MHC-unrestricted nature of this
chimeric receptor allows HIV-specific targeting of both
CD4+ and CD8+ T cells. This may also circumvent
the potential ability of HIV to evade the T-cell immune response
through down-regulation of HLA molecules on the surface of infected
cells.26-28
Preclinical studies of CD4 gene-modified CD8+ T cells
have demonstrated antigen-specific proliferation and cytokine
production, cytolytic activity against HIV-infected T cells, and
inhibition of viral replication in HIV-infected macrophages equivalent
to that seen with naturally occurring HIV-specific CTL
clones.25,29 Preliminary studies of adoptive transfer of ex
vivo-expanded CD4-modified syngeneic CD8+ T cells in
HIV-infected twin pairs demonstrated a rapid decline in gene-marked
cells in the blood, suggesting a lack of HIV-specific CD4+
T-cell help to maintain the transferred CTLs.30 Recent
advances in ex vivo culture methods have allowed us to develop an
efficient ex vivo T-cell stimulation and gene transfer system that
yields mixed populations of gene-modified CD4+ and
CD8+ T cells.31 We now report the data from a
phase II clinical trial of co-infusion of autologous CD4-modified
CD4+ and CD8+ T cells administered with or
without exogenous interleukin-2 (IL-2) in 24 HIV-infected patients with
detectable viral loads.
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Patients and methods |
Subjects
HIV-seropositive individuals over the age of 13 years on stable antiretroviral therapy for more than 8 weeks with viral loads of 1000 to 100 000 copies/mL and CD4 counts
greater than 50/µL were enrolled in this study between May and
December 1997. Informed consent was obtained in accordance with
institutional review board guidelines. Patients were excluded for
significant comorbid illness, active opportunistic infection or
malignancy, or recent history of treatment with immunomodulatory agents.
Vector production
The CD4 retroviral vector rkat4.2SVGF3e is a variant of
the previously described rkat43.3F3 vector and was constructed using the pBR322 plasmid and a murine maloney leukemia virus (MMLV) backbone.32 This vector codes for a chimeric receptor gene
composed of the extracellular and transmembrane domains of human CD4
linked to the cytoplasmic domain of the CD3 T-cell receptor chain. The vector titer on NIH 3T3 cells ranged from 2 to
15 × 106 viruses/mL. Master and working cell banks
and vector lots all tested negative for replication competent
retrovirus (RCR) by supernatant amplification and cocultivation on
Mus dunni cells.33
T-cell processing
Lymphapheresis was performed locally using an automated cell
separator (Cobe Spectra or CS-3000, Lakewood, CO) with processing of 6 to 10 L of blood to achieve a minimum of 5 × 109
peripheral blood mononuclear cells (PBMCs). Apheresis products were
shipped to Cell Genesys, Foster City, CA, at room temperature and
processed within 24 hours of receipt. PBMCs were isolated by density
gradient separation using a Stericell device (Haemonetics, Braintree,
MA). Recovered cells were stimulated using magnetic beads (Dynal, Oslo,
Norway) coated with anti-CD3 (OKT3) and anti-CD28 (monoclonal antibody
9.3), at a bead to cell ratio of 3:1, in serum-free AIM-V medium
(Gibco, Long Island, NY) containing IL-2 (200 IU/mL; Chiron,
Emeryville, CA) and antiretroviral agents (1 µmol/L zidovudine,
Burroughs Wellcome, Research Triangle, NC; 10 µmol/L didanosine,
Bristol Myers Squibb, Princeton, NJ; and 500 µmol/L ritonavir, Abbott
Laboratories, Chicago, IL). On day 3, the beads were removed using a
Maxsep magnetic bead separator (Baxter, Roundlake, IL) and resuspended
in AIM-V medium with IL-2 and ritonavir. Transduction with CD4
retroviral supernatant at a multiplicity of infection of 2 viral
particles per cell was performed on days 5 and 7 in AIM-V medium
containing IL-2, ritonavir, and polybrene (Aldrich, St Louis, MO).
Supernatant was removed by centrifugation using a SteriCell harvester,
and cell expansion in AIM-V medium with IL-2, zidovudine, didanosine,
and ritonavir continued until the target cell dose of
3 × 1010 was obtained (days 10-17). The final
T-cell products were cryopreserved in 50-mL bags containing
6 × 109 cells/bag in Plasmalyte-A (Baxter IV
Systems, Roundlake, IL) with 10% dimethylsulfoxide (Sigma, St Louis,
MO), 1% dextran-40 (Baxter IV Systems), and 5% human serum albumin
(Alpha Therapeutics, Los Angeles, CA), and stored in liquid nitrogen.
Final T-cell products were tested for viability (by trypan blue
exclusion), sterility, Mycoplasma, transduction efficiency
(CD4 presence by polymerase chain reaction [PCR]), HIV replication
(p24 enzyme-linked immunosorbent assay; NEN Life Sciences, Boston, MA),
RCR (cocultivation on M dunni cells), and HIV-specific CTL
activity. Cytolytic activity was assessed in a standard cytotoxic
T-lymphocyte assay with 293 cells (human embryonic kidney line)
expressing HIV env as cytolytic targets.25
Phenotypic analysis was performed using antibodies to CD3,
CD4, CD8, CD28 (Coulter, Miami, FL), CD25 (Caltag, Burlingame, CA), and CD62L (Pharmingen, San Diego, CA). Cryopreserved T
cells were thawed in a 37°C water bath at the patient's
bedside and infused directly through a peripheral intravenous catheter
over 5 to 10 minutes per bag.
Treatment, toxicity, and response evaluation
Subjects were randomized to receive a single intravenous infusion of
2 to 3 × 1010 CD4 gene-modified T cells
administered with or without exogenous IL-2. IL-2 was administered as a
5-day continuous intravenous infusion at a dose of 6 million IU/24
hours, beginning 4 hours before the T-cell infusion. All subjects were followed for at least 8 weeks after the T-cell infusion and monitored for toxicity, changes in plasma HIV viral load (ultrasensitive Roche
Amplicor kit, sensitivity 40 copies/mL), HIV proviral DNA (HIV DNA PCR;
Specialty Labs, Los Angeles, CA), CD4+ T-cell count, and
gene-modified T-cell persistence in peripheral blood (CD4 DNA PCR;
Specialty Labs). A subset of 5 patients was monitored for rectal
mucosa-associated viral burden and tissue trafficking of CD4 T
cells. Follow-up was extended to 1 year in 18 of 24 subjects.
CD4 T-cell survival and gene expression
CD4 DNA PCR.
PBMCs were isolated by standard Ficoll gradient separation. In a subset
of patients, immunoselection for CD4+ and CD8+
Tcells was performed before PCR analysis. Immunoselection of CD8+ T cells from whole blood was performed by first
depleting monocytes using anti-CD14-coated beads (Dynal) followed by
positive selection with anti-CD8-coated beads (> 95% purity).
Immunoselection for CD4+ T cells was performed by first
depleting monocytes and CD8+ T cells from the samples using
anti-CD14- and anti-CD8-coated beads followed by positive selection
with anti-CD4-coated beads (> 95% purity). DNA was recovered by
phenol-chloroform extraction and ethanol precipitation. Quantitation
of globin and CD4 copy number was performed by real-time PCR on an
ABI Prizm 7700 instrument (Taqman PCR; Perkin-Elmer Applied Biosystems,
Foster City, CA). Primers for CD4 PCR amplify a region spanning and the 3' untranslated region and are as follows: forward
primer, 5'-ACC CGG TTC ACT CTT CTC AG-3'; reverse primer,
5'-ACA GGT GGG GTC TTT CAT TC-3'; and internal probe,
5'-(FAM) CAC AGA CTG TTG TCC CTG CAC TCT (TAMRA)-3'. PCR
reactions were carried out with the Taqman PCR reagent kit (Perkin-Elmer Applied Biosystems). Amplification was performed for 45 cycles (initial activation: 94°C, 10 minutes; cycling: 94°C, 15 seconds, 57.5°C, 45 seconds). Copy number was determined using
normal PBMC DNA as a beta-globin standard and CD4 PCR product as the
standard for CD4 quantitation. Assay sensitivity was 117 copies/106 cells.
CD4 RNA reverse transcriptase
(RT)-PCR.
Total RNA was isolated from PBMC pellets using TriReagent (Molecular
Research Center, Cincinnati, OH) following the manufacturer's protocol; cDNA was prepared using Superscript II reverse transcriptase (Life Technologies, Long Island, NY). Samples were incubated at 48°C for 90 minutes and then digested with RNase H at 37°C. RNA isolated from PBMC pellets from HIV-negative human donors was used as a control. PCR amplifications were performed using TaqMan technology, as described earlier. CD4 PCR product was used to generate a CD4 standard curve. To normalize results for the
efficiency of mRNA isolation, we amplified reduced
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
in a separate Taqman PCR reaction and used it to generate a
standard reference series. Data analyses for CD4 and GAPDH
amplifications were performed using the Sequence Detection System
software (version 1.6.3) default settings.
HIV proviral DNA assay
DNA was isolated from PBMCs, and quantification of globin and HIV-1
proviral DNA copy number was performed by Taqman PCR, as described for
CD4 DNA PCR. Primers sequences recognized HIV gag and were
as follows: forward primer, 5'-CTC TAA GAG CCG AGC AAG CTT
C-3'; reverse primer, 5'-GGT CCT CCT ACT
CCC TGA CAT-3'; and internal probe, 5'-(FAM)-AAG CAT TGG
GAC CAG CGG CTA CAC T-(TAMRA)-3'. Amplification was performed for
50 cycles with the same cycling conditions as for CD4 DNA PCR.
Plasmid BH1034 was used as a standard for HIV-1 DNA copy
number quantification. BH10 contains a replication-defective copy of
the HIV-1 subtype B isolate HxB2. Assay sensitivity was 35 copies/106 cells.
Replication-competent retrovirus (RCR) testing in patients
Analysis for RCR was performed using Taqman PCR.
Genomic DNA was isolated from PBMCs using the Puregene kit (Gentra
Systems, Minneapolis, MN) following the manufacturer's protocol. PCR
primers S-RCR-F1 (5'-GGG ACA CGG GAT GCT CTA AA) and S-RCR-R1
(5'-GGA AGG AAT TGG ATA CTT TGG AGA) amplify a 70-bp sequence in
the envelope region of the amphotropic murine leukemia virus. AmpErase
(UNG. 0.5U) was included in the PCR reaction. PCR amplifications were performed in an ABI Prizm 7700 instrument using the following cycling
conditions: 50°C × 2 minutes, 95°C × 10 minutes,
95°C × 15 seconds, and 60°C × 1 minute (40 cycles).
Data analysis was performed using the Sequence Detection System
software, as described previously. A 466-bp PCR product derived from
the MMLV plasmid pkat2Ampac.Utd was used to generate a standard curve
in triplicate (1-1000 template copies) for assay quantitation. Assay
sensitivity was 7 copies/106 cells.
Rectal biopsy substudy
Patients enrolled at the University of California, Los Angeles were
eligible for a substudy to analyze serial biopsies of rectal mucosa for
viral burden and the presence of CD4-modified T cells.
Biopsy acquisition.
Flexible sigmoidoscopy was performed 1 week before T-cell infusion and
at days 3, 7, and 14 after infusion. At each procedure, 10 biopsies
(large-cup, 3.3 OD, 8 mm span; Microvasive) were obtained circumferentially at a standard level of 30 cm. Each biopsy was approximately 2 to 3 µL. Six biopsy specimens were immediately frozen
in liquid nitrogen and stored at 80°C. Four specimens were
placed in RPMI medium for subsequent isolation of mucosal mononuclear cells.
Isolation of mucosal mononuclear cells for CD4
analysis.
Four biopsy specimens in RPMI 1640 medium were transferred into a
sterile petri dish containing
Ca2+/Mg2+-free
phosphate-buffered saline (PBS) supplemented with 50 µmol/L 2-mercaptoethanol and 100 mmol/L EDTA, and teased apart using two 18G
needles. The dispersed tissue was incubated at 37°C in a shaking
water bath for 20 minutes, washed twice with PBS, resuspended in 0.1 mg/mL of collagenase/dispase (Boehringer Mannheim, Indianapolis, IN) in
RPMI 1640, and further incubated at 37°C with shaking for 60 minutes. The digested tissue was repeatedly aspirated through an 18G
followed by a 20G needle and filtered through a 70-µ cell strainer to
yield a single cell suspension. Mononuclear cells were enumerated
visually, and viability was assessed by trypan blue exclusion. Absolute
cell numbers were confirmed by flow cytometry with the use of Trucount
beads (Becton Dickinson, San Jose, CA). These techniques yielded an
average of 0.2 to 5.0 × 105 CD45+
mononuclear cells, 1.0 to 4.0 × 104
CD4+ T cells, and 1.0 to 8.0 × 104
CD8+ T cells per 4 biopsies.35
HIV RNA analysis of bulk rectal tissue.
Biopsies from each patient were homogenized (Powergen homogenizer;
Fisher Scientific, Pittsburgh, PA) and Trizol-extracted (Gibco), with
separation of RNA and DNA phases to ensure that RNA for viral load
assays and DNA for CD4 analysis were from the same biopsy. RNA was
further extracted with the Rneasy kit (Qiagen, Valencia, CA). All
analyses were performed on 2 biopsy specimens extracted independently
and run in duplicate.36,37 RT-PCR for HIV RNA was performed
using the Thermostable rTth Reverse Transcriptase RNA PCR kit
(Perkin-Elmer) with oligonucleotide primer pairs 667/AA55 specific for
the R/U5 region of HIV LTR RNA and was designed to
capture both unspliced and multiply spliced HIV RNA.38
Primer 667 was radiolabeled using T4 kinase with 32P. A linear standard curve was generated
by amplifying the in vitro synthesized HIV LTR RNA sequences diluted in
seronegative tissue RNA. Percentage RNA recovery was estimated using
seronegative frozen biopsies with known quantities of a synthesized
140-bp sequence recognized by the 667/AA55 primers and was greater than 95%. PCR products were quantified using the Ambis Image Analysis System. Sensitivity was 10 copies/µg RNA.
CD4 PCR assay.
DNA was extracted from frozen rectal biopsies and mucosal mononuclear
cells using Trizol reagent, as described earlier, with DNA subsequently
isolated by ethanol precipitation with 2 washes in 0.1 mol/L sodium
citrate/10% ethanol buffer. Isolated DNA was transferred to Cell
Genesys for amplification of CD4 DNA.
Statistical analysis
Treatment groups were compared for differences in absolute levels
and changes from baseline for each efficacy end point with a 2-sample
Student t test and a 2-sample Wilcoxon test, and adjustment was
made for multiple correlated comparisons. Undetectable values for
efficacy end points were set to 50% of the detection limit for the
purposes of analysis. For change from baseline
analyses, an average of 2 preinfusion values (week 1 and week 0)
was used to calculate the baseline. Adverse events were coded using the fifth edition of the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART).
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Results |
Patient characteristics and treatment
Twenty-five patients were enrolled in the study between May 29, 1997, and December 3, 1997. One patient was removed from the study
before cell infusion because of failure of T-cell processing. Twenty-four patients received cell infusions and completed 8 weeks of
follow-up. Eleven patients were randomized to receive a single infusion
of 2 to 3 × 1010 CD4-modified T cells plus IL-2
(6 million IU/24 hours by continuous intravenous infusion × 5 days), and 13 were randomized to receive CD4-modified T cells alone. Table 1 lists the patient demographics and
baseline laboratory data for the 24 treated patients. The majority of
patients were male, white, and 30 to 50 years of age, and had an
excellent performance status. Twenty (83%) had been diagnosed with HIV
infection more than 3 years before enrollment. Mean viral load at
enrollment was 20 137 copies/mL and mean CD4+ T-cell count
was 332/µL. There were no statistically significant differences
between the treatment groups for any screening covariates. One patient
was taking no antiretroviral drugs and 23 (96%) were taking stable
antiretroviral medications from at least 8 weeks before cell infusion
through 8 weeks of follow-up, with 1 exception (switch from
lamivudine/indinavir to saquinavir/nelfinavir 36 days after infusion).
Twenty (83%) were taking protease inhibitors, 6 (25%) were taking
non-nucleoside reverse transcriptase inhibitors, and 7 (29%) were
taking hydroxyurea.
Characteristics of CD4 T-cell products
CD4-modified T cells were successfully processed and released in
24 of 25 patients. A total of 27 lymphapheresis products were
processed, and the T-cell growth curves are shown in Figure 1. Two patients underwent repeat apheresis
because of initial culture failure, and 1 was successfully processed
the second time. A common feature of the 3 failed cultures was a low
lymphocyte content of the incoming apheresis products.
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| Fig 1.
T-cell growth curves of 27 autologous CD4-modified
CD4+ and CD8+ T-cell products in 25 enrolled subjects.
The minimum starting cell count was 5 × 109.
Transduction with CD4 retroviral supernatant occurred on days 5 and
7, followed by expansion in serum-free medium containing IL-2 (200 IU/mL) and antiretroviral agents until reaching the target cell dose of
3 × 1010 cells (horizontal line on chart). Cell
processing was successful in 24 of 25 subjects (the 2 failed cultures
are depicted with the dashed lines). Both patients underwent repeat
lymphapheresis, and 1 was successfully processed the second
time.
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Characteristics of the 24 successful T-cell products are shown in Table
2. Cells were expanded ex vivo for an
average of 13 days, and all cultures were completed within 17 days.
This yielded a mean of 4.2 × 1010 cells (range,
1.9-6.8 × 1010 cells). Average transduction
efficiency as measured by PCR for CD4 was 19% and ranged from 5%
to 57%. The average composition of the final T-cell products yielded a
ratio of CD8 to CD4 T cells of approximately 3:1. Similar expansion of
T cells from HIV-uninfected donors yields a higher proportion of
CD4+ T cells with a ratio of approximately 1:1 (data not
shown). Phenotypic analysis by flow cytometry revealed a high
proportion of cells expressing CD62L and CD28, with intermediate
expression of CD25. There was no clear association between the
proportion of CD4+ T cells in the final product or
transduction efficiency and patterns of in vivo survival of the
gene-modified cells.
Toxicity
There were no serious adverse events reported, and T-cell infusions
overall were associated with minimal toxicity (Table
3). Grade 3 or 4 adverse events were
predominantly associated with IL-2 infusion. The most common toxicities
of grade 2 or higher seen in the cells-only arm were fever, chills,
rash, and sinusitis (each seen in 2 patients). Shift-table analyses of
safety laboratory parameters demonstrated an increase in white blood
cells and eosinophils in all patients in the cells + IL-2 group and in
8 of 11 in the cells-only group during the first week following
infusion. Cholesterol decreased in 20 of 22 patients and lactate
dehydrogenase increased in 16 of 17 in both groups
combined. All 24 subjects tested negative for RCR by PCR analysis for
MLV env up to 1 year after infusion.
CD4-modified T-cell survival in peripheral blood
CD4-modified T cells were detected by DNA PCR for the CD4 gene
in the peripheral blood of all patients following infusion. Sustained
mean levels of 4.0 to 4.4 log copies/106 cells (1% to 3%
of PBMCs) were detected at all time points after infusion from day 3 through week 8 in both treatment arms (Figure 2A). Extended follow-up through 12 months
in 18 patients demonstrated sustained persistence of CD4-modified T
cells in the blood of 17 of 18, with mean values of 3.8 log copies
(cells + IL-2) and 3.1 log copies (cells only) at 6 months, and 3.1 log
copies (cells + IL-2) and 2.9 log copies (cells only) at 12 months.
There were no statistically significant differences in survival of
gene-modified T cells between the treatment arms. Fractionation of
blood T cells was performed in 6 patients and demonstrated survival of
both gene-modified CD4+ and CD8+ T cells in all
patients analyzed (Figure 2B). In addition, CD4 RNA RT-PCR analysis
was performed in 6 patients and confirmed relatively stable gene
expression for at least 16 weeks (Figure 2C).
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| Fig 2.
CD4-modified T-cell survival and gene expression in
peripheral blood mononuclear cells (PBMCs).
Gene-modified T-cell infusion was administered at week 0, and patients
were followed for at least 8 weeks after infusion. (A) Survival of
CD4-modified T cells in peripheral blood as measured by quantitative
DNA PCR analysis for CD4 in the cells only ( ) and cells + IL-2
( ) treatment cohorts through 8 weeks of follow-up. There were no
statistically significant differences between cohorts at any time point
after infusion (P = .35, week 2; P = .49, week 4;
P = .93, week 8). (B) Immunoselected
populations of CD4+ () and CD8+ ( ) T
cells in peripheral blood were analyzed for the presence of CD4 by
DNA PCR and compared with the presence of CD4 in the bulk population
(). The results confirmed the persistence of both gene-modified
CD4+ and CD8+ T cells in 6 of 6 patients
analyzed for at least 8 weeks. Results of 1 representative patient
(J233) out of a total of 6 analyzed are shown. (C) Persistent,
relatively stable expression of the CD4 gene was confirmed for at
least 16 weeks by CD4 RNA RT-PCR analysis of PBMCs in 6 patients
(G251, T253, Y254, Q202, Y292, and J233).
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Antiviral activity of CD4 T cells
Antiviral activity of CD4 T-cell infusions administered with or
without IL-2 was assessed through monitoring of plasma HIV viral load,
blood HIV proviral DNA, CD4+ T-cell counts, and analysis of
HIV reservoirs in rectal mucosa. A change in plasma viral load of
greater than 0.5 log is unlikely to be explained by inherent biologic
or assay variability and is accepted as a clinically relevant change in
the level of plasma HIV RNA.39 There was no change in mean
plasma viral load greater than 0.5 log at any time point after infusion
in either treatment arm, and differences between arms were not
statistically significant (Figure 3A). Two
patients (8%; 95% confidence interval [CI], 1% to 27%)
experienced a viral load decrease of 0.5 log or greater at week 2, 3 patients (23%; 95% CI, 3% to 34%) at week 4, and 1 patient (4%;
95% CI, 0 to 21%) at week 8, suggesting a transient antiviral effect
of gene-modified T cells in a subset of patients. There was no
statistically significant difference in the proportion of patients
experiencing a 0.5 log or greater decrease in viral load between the
treatment groups.
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| Fig 3.
Antiviral activity and change in CD4+
T-cell count following CD4-modified T-cell infusion.
Baseline values represent an average of those at week 1 and week
0. T-cell infusion occurred at week 0, and postinfusion analyses were
performed on days 1, 2, and 3 and weeks 2, 4, and 8. Scr1 and 2 represent the 2 screening values performed approximately 8 weeks before
cell infusion. (A) Mean change from baseline in plasma HIV RNA (Roche
Amplicor kit; sensitivity 40 copies/mL). There were no statistically
significant differences between the cells only () and cells + IL-2
() cohorts at any time point after infusion (P = .87, week
2; P = .70, week 4; P = .86, week 8). (B) Mean
change from baseline in blood HIV proviral DNA (DNA PCR for HIV
gag, Specialty Labs; sensitivity 35 copies/106
cells). There were no statistically significant differences between the
cells only () and cells + IL-2 () cohorts at any time
point after infusion (P = .84, week 2; P = .94,
week 4; P = .89, week 8). (C) Mean change from baseline in
CD4+ T-cell count. There was a trend toward a greater
increase in CD4+ T-cell counts after infusion in the cells + IL-2 arm () compared with the cells-only arm ()
(P = .04, week 2; P = .14, week 4;
P = .10, week 8).
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The mean change from baseline in blood HIV proviral DNA did not exceed
0.10 log in either treatment arm during the 8-week observation (Figure
3B). However, the HIV proviral DNA assay used in this trial measures
total HIV DNA including integrated, unintegrated, and
replication-incompetent copies and may not, therefore, accurately reflect changes in replication-competent HIV proviral DNA.
Figure 3C shows the mean change from baseline in the CD4+
cell counts in the 2 treatment arms. CD4+ counts decreased
transiently in the cells + IL-2 arm during the first 3 days following
T-cell infusion (concomitant with IL-2 infusion), which is consistent
with transient lymphopenia induced by IL-2 (C. Lane, personal
communication). There was an increase in CD4+ counts in
both treatment arms at week 2; however, this increase was sustained
only in the cells + IL-2 arm at week 8, with a mean increase of 73 cells/µL (95% CI, 19-127). This is consistent with the expected
effect of IL-2 alone.40 The difference between treatment
arms at week 8 was of borderline statistical significance (P = .10, nontransformed CD4+ cell count;
P = .07, log-transformed CD4+ cell count).
CD4 T-cell trafficking and activity against gut-associated
HIV reservoirs
The gut-associated lymphoid tissue (GALT) serves as an important and
accessible tissue reservoir of HIV-infected cells. Five of 6 eligible
patients (3 receiving cells only; 2 receiving cells + IL-2)
participated in a substudy to investigate the trafficking and antiviral
activity of CD4-modified T cells against rectal tissue reservoirs
of HIV. All 5 completed the planned rectal biopsy procedures
(week 1; days 3, 7, and 14), and there were no
procedure-related adverse events.
CD4 signal was measured in PBMCs, bulk tissue, and isolated lamina
propria lymphocytes (LPLs) by PCR (Table
4). CD4 signal was consistently detected
in whole biopsy tissue samples in all patients at all time points (mean
2.6 log copies/106 tissue cells). The mean
CD4 signal in blood for these 5 subjects over the 14-day observation
period was 4.1 log copies/106 PBMCs. Long-term follow-up
demonstrated persistent CD4 signal in rectal tissue in 2 of 3 subjects at 1 year. CD4 signal in the rectal biopsy specimens was
consistently lower than that in peripheral blood. This could reflect
the difference in the denominator (fractionated lymphocytes versus bulk
tissue), the kinetics of trafficking to the gut mucosa, or loss of
signal associated with sample processing. All subjects showed an
increase in CD4 signal in bulk rectal tissue from day 3 to day 7. Detection of CD4 in isolated LPLs was less reproducible; however,
signal was detected in LPLs from every patient at 1 or more time points
after infusion. These data are consistent with the trafficking of
gene-modified T cells to rectal lymphoid tissue in all 5 patients with
maintenance of CD4 signal for at least 14 days.
Rectal tissue-associated HIV RNA load was measured by HIV RNA RT-PCR
analysis (Table 4). The mean tissue viral load decreased from the
preinfusion value by at least 0.5 log copies/µg RNA at all time
points after infusion. Four of 5 patients showed a 0.5 log or greater
decrease at 2 of 3 time points, and 2 of 5 showed a 0.5 log or greater
decrease at all measured time points. Three patients had a 1.0 log or
greater decrease in tissue HIV RNA at 1 or more time points after
infusion. These preliminary data suggest antiviral activity of
CD4-modified T cells against gut-associated reservoirs of HIV. There
were too few patients to assess the impact of concomitant IL-2
administration; however, both tissue trafficking and antiviral activity
were demonstrated in patients in both treatment arms. The duration of
this effect and verification of these results will require further study.
Measurement of plasma-associated HIV RNA did not follow the same trend
as seen in HIV RNA in the gut tissue in these 5 patients (Table 4).
There was no significant mean change (> 0.5 log copies/mL) in plasma
viral load during the 14-day observation period; however, 1 of 5 patients showed an isolated decrease in plasma viral load of at least
0.5 log at day 7.
| |
Discussion |
We have developed a rapid T-cell manufacturing process that reliably
yields 2 to 3 × 1010 gene-modified T cells within 2 weeks. This process was successful in 24 of 25 enrolled patients and
yielded a mixed population of CD4+ and CD8+ T
cells with an activated cell phenotype (high expression of CD28, CD25,
and CD62L). Bulk transduction efficiency in this trial was
approximately 20% and, with further processing improvements, has been
increased to 40%.41 This high-level transduction
eliminates the need for posttransduction purification of gene-modified
cells, allowing for the brief period of ex vivo expansion.
CD4-modified CD4+ and CD8+ T cells persisted
in the blood of all patients at high levels (1% to 3% of PBMCs)
throughout the 8-week observation period, and extended follow-up
demonstrated maintained survival for at least 1 year in 17 of 18 subjects (0.1% of PBMCs). This long-term persistence is in marked
contrast to our previous experience with infusion of syngeneic
CD4-modified CD8+ T cells in HIV-infected twin pairs as
well as the previously published experience with adoptive T-cell
therapy in HIV infection reported by other groups. In the syngeneic
twin study, gene-modified CD8+ T cells were purified and
expanded in IL-2 for a mean of 62 days. Peak circulating levels of
CD4-modified T cells of 1% to 10% of PBMCs were observed at 48 hours, followed by a 2- to 3-log decrease within 8 weeks of adoptive
transfer (manuscript submitted). Infusion of autologous
neo-marked HIV gag-specific CD8+ T cells by
another group showed similarly poor survival patterns, with
neo-marked cells constituting 2% to 3.5% of CD8+
T cells in blood 1 day after infusion (3.3 × 109
cells/m2), and with a rapid decline to fewer than 5 cells
per 106 PBMCs by 3 weeks.24 The most likely
explanation for the improved T-cell survival, independent of exogenous
IL-2, seen in this trial is a helper effect conferred by
CD4-modified CD4+ T cells. However, changes in ex vivo
T-cell stimulation and/or the shorter total duration of ex vivo cell
culture (mean of 13 versus 62 days) may also have played a role. This
high-level, sustained, IL-2-independent survival of gene-modified
autologous T cells validates this approach as a platform technology for
gene delivery.
The T-cell culture method used in this study provided costimulation of
CD4+ and CD8+ T cells by antibodies to CD3 and
CD28 co-immobilized on magnetic beads. This differs from the original
twin study in which single signal T-cell stimulation of purified
CD8+ T cells with anti-CD3 and high-dose IL-2 was employed.
This new cell culture method allows ex vivo proliferation of
CD4+ T cells and renders CD4+ T cells resistant
to infection with M-tropic strains of HIV through down-regulation of
the HIV fusion coreceptor CCR5.31,42 In preclinical
experiments at Cell Genesys, this method generated T cells that were
resistant to antigen-induced apoptosis, whereas CD3/IL-2-stimulated
cells died rapidly following repeated rounds of stimulation through the
CD4 receptor. Furthermore, this new process generated
CD4+ T cells capable of proliferation and IL-2 secretion
upon CD4 receptor engagement. Gene-modified CD4+ and
CD8+ T cells showed equivalent cytolytic activity against
HIV env-bearing targets and equivalent potency in
suppression of HIV replication in infected T-cell cultures as
CD8+ T cells alone cultured using
anti-CD3/IL-2.43 The markedly improved, sustained,
high-level in vivo survival of both gene-modified CD4+ and
CD8+ T cells in this trial is consistent with these
preclinical observations and suggests that mixed CD4+
and CD8+ T-cell populations provide HIV-specific
T-helper function.
Preliminary evidence of antiviral activity of CD4-modified
CD4+ and CD8+ T cells was observed in this
trial. Although there was no significant mean change, several patients
experienced a plasma viral load decrease of greater than 0.5 log on at
least 1 occasion, suggesting a transient antiviral effect in a subset
of patients. Furthermore, a greater than 0.5 log mean decrease in
rectal mucosa-associated HIV RNA was detected from days 3 through 14 in a subset of 5 patients who underwent serial rectal biopsies,
suggesting antiviral activity against this important tissue reservoir
of HIV. This antiviral effect correlated with detection of
CD4-modified T cells in the rectal tissue of all 5 patients
throughout the 14-day monitoring period, with increasing signal from
day 3 to day 7. Possible explanations for the discordance between
changes in gut and plasma viral loads include true compartment
differences in the activity of gene-modified T cells, differences in
the kinetics of activity between the compartments, or insufficient
magnitude of effect in the gut compartment to be reflected in plasma
viral load. Expression of the  7 integrin subfamily of adhesion
molecules has been shown to be critically important for lymphocyte
trafficking to GALT, and the formation of GALT is severely impaired in
mice deficient for this receptor family.44 Subsequent
experiments have confirmed the expression of  47 on
CD4-modified T cells at the end of ex vivo cell culture (unpublished
data). The gastrointestinal tract contains most of the lymphoid tissue
in the body and has been shown to be a major target for SIV replication
and CD4+ T-cell loss in early SIV replication following
intravenous inoculation in Rhesus macaques.45 Therefore,
demonstration of antiviral activity against this tissue reservoir of
HIV is likely to be important in the evaluation of novel agents. We did
not investigate lymph node trafficking of CD4-modified T cells in
this study; however, another group has demonstrated accumulation of
neo-marked HIV-specific CD8+ T cells adjacent to
infected CD4+ T cells in lymph nodes, confirming the
ability of adoptively transferred lymphocytes to migrate to this
lymphoid reservoir.24
The results of this clinical trial validate the feasibility of adoptive
immunotherapy of HIV infection with genetically modified, MHC-unrestricted, polyclonal T cells bearing chimeric HIV
env-specific immune receptors and provide preliminary evidence
of tissue homing and antiviral activity against tissue reservoirs of
HIV. Our belief, based on the experience in adoptive T-cell
immunotherapy of cancer, is that this therapy is most likely to
demonstrate convincing clinical efficacy in subjects with minimal viral
burden. Among patients receiving donor lymphocyte infusions for
relapsed chronic myelogenous leukemia following bone marrow
transplantation, remissions are most frequent in patients with
cytogenetic or early hematologic relapse and less common in patients
with advanced disease.46 We have therefore initiated a
randomized controlled clinical trial of CD4-modified versus
-unmodified T cells in 40 HIV-infected subjects with undetectable
plasma viral loads taking combination antiretroviral therapy to measure
the efficacy of this gene therapy against residual blood and tissue
reservoirs of HIV.
| |
Acknowledgments |
We would like to acknowledge the following people for their
contributions to this study: Judith Carden, Alison Leiblein, Julie Elliott, Dorie Heeren, Walter Howard, Kathleen Shea, Virginia Waite,
Janet Wittes, Lynne Fitch, and David Broad.
| |
Footnotes |
Submitted December 8, 1999; accepted March 12, 2000.
Supported by Cell Genesys Inc in collaboration with Hoechst
Marion Roussel. P.A.A. was supported in part by Mucosal Immunology CORE
Grant no. AI28697, University of California, Los Angeles. This work was
carried out in part in the General Clinical Research Centers at the
following institutions: University of California, Los Angeles
(RR-00865), San Francisco General Hospital (5-MO1-RR00083-37), and
University of Colorado Health Science Center
(RR-00051).
Reprints: Kristen M. Hege, Cell Genesys, Inc, 342 Lakeside Dr,
Foster City, CA 94404; e-mail: kristenh{at}cellgenesys.com.
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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Top
Abstract
Introduction