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PLENARY PAPER
From the Division of Hematology/Oncology, Department of
Medicine, and the UCSD Human Gene Therapy Program, University of
California-San Diego, La Jolla, CA; Immunogenex, Inc, La Jolla, CA; and
Molecular Medicine/LLC, UCSD, La Jolla, CA.
Chronic lymphocytic leukemia (CLL) cells can be made to express
recombinant CD40-ligand (CD154) by transduction with a
replication-defective adenovirus vector (Ad-CD154).
Ad-CD154-transduced and bystander leukemia cells become highly
effective antigen-presenting cells that can induce CLL-specific
autologous cytotoxic T lymphocytes in vitro. This study investigated
the immunologic and clinical responses to infusion of autologous
Ad-CD154-CLL cells in patients with CLL. After a one-time bolus
infusion of autologous Ad-CD154-transduced leukemia cells, there was
increased or de novo expression of immune accessory molecules on
bystander, noninfected CLL cells in vivo. Treated patients also
developed high plasma levels of interleukin-12 and interferon- Chronic lymphocytic leukemia (CLL) is the most
common adult leukemia in the United States.1 It is
characterized by progressive accumulation of well-differentiated
malignant monoclonal B lymphocytes in blood, lymph nodes, liver,
spleen, and marrow.2 Progression of the disease is
typified by increases in blood lymphocyte count; increases in the size
of lymph nodes, liver, and spleen; and advancing anemia and
thrombocytopenia. Although chemotherapy may palliate symptoms, there is
no established cure. This necessitates development of new treatments
with a different mechanism of action.
Several features of this disease suggest that immune-based strategies
may have therapeutic potential. CLL B cells are slowly dividing,
monoclonal B cells that express differentiation antigens, major
histocompatibility complex (MHC) class I and II molecules, and surface
immunoglobulin (Ig).2,3 The Ig molecules expressed by the
CLL B cells have features that distinguish them from the Ig molecules
expressed by normal, nonmalignant B cells, even when encoded by
nonmutated Ig variable region genes.4-6 Also, complex cytogenetic abnormalities frequently accumulate over time in CLL B
cells.2,3,7,8 These genetic differences could result in
CLL-specific antigens that may be recognized by the host immune system.
However, despite expressing high levels of MHC class I and II
molecules, CLL B cells are ineffective antigen-presenting cells (APCs).
Indeed, CLL B cells are unable to stimulate even normal allogeneic T
cells in a mixed lymphocyte reaction (MLR).9-13 This is
due in part to their lack of important costimulatory surface molecules,
such as CD80 and CD86, which are necessary for efficient T-cell
activation.9,14-16
We found that activated T cells could induce CLL B cells to become
effective APCs.9 This effect was mediated largely by the
ligand for CD40, designated CD154. CD154 is a type II membrane glycoprotein that is expressed transiently by activated
CD4+ T cells.17 Normally, CD154 engages CD40
on potential APCs, including B cells, monocytes, and dendritic cells,
and induces expression of accessory surface molecules that are
important in cognate costimulatory cell-cell interactions. CLL B cells
express CD40 and also are induced to express de novo or enhanced
amounts of CD54, CD70, CD80, CD86, and CD95, following CD40 ligation in vitro. Such phenotypic changes allow these cells to function as effective stimulators of allogeneic or autologous T cells in a mixed
lymphocyte culture.18 Conceivably, such activated
neoplastic B cells also may present tumor-associated antigens to
autologous T cells, allowing for the immune rejection of tumor, as
recently observed in animal models of B-cell
lymphoma.19,20
The CLL B cells can be transduced in vitro with high-titer,
replication-defective adenovirus vector to express high levels of
transgene.21 A replication-defective adenovirus vector was constructed using serotype 5 adenovirus in which the E1 region of the
virus genome was replaced with the gene encoding murine CD154 flanked
in the 5' direction with a heterologous cytomegalovirus (CMV) promoter,
and in the 3' direction with a bovine polyadenylation signal
(Ad-CD154). Substantial numbers of CLL B cells can be transduced in
vitro to express the CD154 transgene using this adenovirus vector.18 Expression of CD154 transgene by CLL B cells
simulates exposure of these cells to activated T cells and induces
leukemia cell expression of immune costimulatory surface molecules. In addition, CLL B cells that express CD154 can induce noninfected, bystander CLL B cells to express such surface molecules. When tested in
vitro, Ad-CD154-infected CLL B cells, but not CLL cells infected with an adenovirus vector encoding an irrelevant transgene, could stimulate allogeneic as well as autologous T cells in an MLR,
thereby inducing T-cell proliferation and production of interferon- Patients and CLL B-cell transduction
After providing informed consent, patients underwent leukapheresis.
Blood mononuclear cells were isolated via density gradient centrifugation and then infected for 24 hours with high-titer replication-defective Ad-CD154 in a Good Manufacturing Practice (GMP)
facility, as described.21 Following infection, the cells were washed extensively to remove free virus and viably frozen in 10%
dimethylsulfoxide (DMSO) for storage in liquid nitrogen. An aliquot of
cells was taken before freezing for bacterial culture to ensure
sterility. On the day of treatment, the transduced cells were thawed
and washed before intravenous infusion. The autologous transduced cells
were administered as a single intravenous bolus infusion over
approximately 10 minutes.
Mononuclear cell and T-cell isolation
Reverse transcriptase-polymerase chain reaction Total RNA was isolated from 5 × 106 blood mononuclear cells using RNeasy kit (Quiagen, Chatsworth, CA). First strand complementary DNA (cDNA) was synthesized using 5 µg of total RNA, the Superscript cDNA synthesis kit (GIBCO BRL, Gaithersburg, MD), and oligo-dT primers. The residual RNA was removed with RNAseH and one fourth of the cDNA was used in the polymerase chain reaction (PCR) reaction. The PCR reactions were performed for 30 cycles in 50 µL of 1 × Boehringer Mannheim amplification buffer (Indianapolis, IN), Taq polymerase, and 200 µmol/L of dNTP. Primers for the murine CD154 consisted of the sense primer 5'-GATGAGGATCCTCAAATTG-3' and the antisense primer 5'-GTTTCTAGATCAGAGTTTGAGTAAGCC-3' (nt 346-783); these amplified a 437-bp fragment of the 3' end of the CD154 molecule. One fifth of the amplified PCR product was visualized on a 1% agarose gel by ethidium bromide staining.Enzyme-linked immunosorbent assay for plasma cytokines We measured the levels of plasma cytokines by enzyme-linked immunosorbent assay (ELISA). We added antihuman IFN- , interleukin (IL)-6, IL-1 , IL-4, or tumor necrosis factor (TNF)- capture antibody (PharMingen, San Diego, CA) at 10 µg/mL phosphate buffer (0.1 mol/L Na2HPO4, pH 9.0) to individual wells
of a 96-well EIA/RIA A/2 ELISA plate (Costar, Cambridge, MA). After an
overnight incubation at 4°C, the plates were washed twice with wash
buffer (0.05% Tween 20 in phosphate-buffered saline [PBS]). The
wells then were filled with PBS containing 10% fetal calf serum (FCS)
to block residual protein-binding activity. The plates were washed 4 times with wash buffer and incubated overnight with the patient plasma
samples at 4°C. Recombinant human IFN-, IL-6, IL-1, IL-4, or
TNF- was used for standard curve (1:2 serial dilutions in detection
buffer (10% FCS, 0.05% Tween 20 in PBS). The plates were washed 4 times in wash buffer and incubated for 1 hour at room temperature with biotinylated-mouse antihuman IFN-, IL-6, IL-1, IL-4, or TNF- (PharMingen) 5 µg/mL in detection buffer. The plates were washed twice and treated with Avidin and biotinylated horseradish peroxidase (Elite Vectastain, Vector Laboratories, Burlingame, CA) for 1 hour at
room temperature. IL-2 and IL-12 (p70) concentrations were determined
using OptEIA ELISA kits (PharMingen, La Jolla, CA). After the plates
were washed 5 times, the substrate TMB peroxidase was added (Kirkegaard
& Perry Laboratories, Gaithersburg, MD). The OD at 450 nm was measured
using an ELISA microplate reader (Molecular Devices, Menlo Park, CA)
and cytokine concentrations were extrapolated from the
standard curves.
ELISPOT assay One hundred microliters of anti-IFN- antibody (clone no.
2G1, Endogen, Woburn, MA) at 10 µg/mL in sterile PBS (pH 7.4) was added to each well of a 96-well nitrocellulose plate (Millipore, Bedford, MA). After an overnight incubation at 4°C, the plates were
washed 4 times with sterile PBS. Residual protein-binding sites were
blocked by adding 200 µL/well of PBS containing 1% bovine serum
albumin (BSA) or 10% FCS for a 30-minute or longer incubation at
37°C prior to adding cells. Serial dilutions of autologous T cells
suspended in serum-free AIM-V media were added to separate wells of the
microtiter plate (100 µL/well). A fixed number
(1 × 105 cells/well) of CD40-activated autologous CLL B
stimulator cells was added to wells to give varying
responder/stimulator ratios. The CD40-activated autologous CLL B cells
were prepared from leukapheresis mononuclear cells by culturing the
cells for 48 hours at 37°C either with HeLa cells transfected to
express human CD154 or with their culture supernatant, which contained
approximately 5 ng/mL soluble CD154, as assessed by ELISA. All assay
media contained recombinant IL-2 at a final concentration of 25 U/mL.
The murine anti-HLA mAb W6/32 was added to a final concentration of 10 µg/mL to some of the wells, as indicated. The plates were incubated for 48 hours at 37°C in a 5% CO2 incubator, washed free
of cells 4 times with PBS, and then washed 4 times with PBS containing 0.05% Tween 20. One hundred microliters of biotinylated secondary anti-IFN- antibody (clone B133.5, Endogen) was added to each well
at 1 µg/mL in PBS containing 4% BSA. After 1 hour of incubation at
37°C, the wells were washed 4 times with PBS with 0.05% Tween 20. Then, 100 µL of horseradish peroxidase (HRP)-conjugated Streptavidin at 1:500 concentration in PBS with 0.05% Tween 20 was added to each
well. The plates were incubated for 30 minutes at room temperature. The
wells then were washed 4 times with PBS containing 0.05% Tween 20. Fresh AEC substrate was prepared by dissolving one tablet of
3-amino-9-ethylcarbazol (Sigma) in 5 mL dimethylformamide and then
diluting this into 45 mL of sodium acetate buffer, pH 5.0, that
contains 25 µL of freshly added 30% hydrogen peroxide. Then 100 µL
of AEC substrate was added to each well and the plates were incubated
for 5 minutes or more at room temperature. The substrate solution was
discarded and the plates were rinsed with tap water and air-dried.
After the plates were completely dry, spots were counted for each well
manually with a stereomicroscope.
Infusion of Ad-CD154-transduced autologous CLL B cells Eleven patients with progressive, intermediate or high-risk CLL by the modified Rai criteria were treated (Table 1). Patients received their autologous transduced cells an average of 44 ± 23 days after leukapheresis (range, 10-77 days). Five patients received approximately 3 × 108 (pilot group and group 1), 3 received approximately 1 × 109 (group 2), and 3 received approximately 3 × 109 (group 3) autologous Ad-CD154-transduced CLL cells according to a dose-escalation design (Table 1).Fewer than 8% of the leukemia cells from the first 2 patients (pilot
group) expressed detectable levels of the CD154 transgene, as assessed
by direct monoclonal antibody (mAb) staining and flow cytometric
analysis (Figure 1A). The 2 patients in
the pilot group did not have a discernible clinical response to the
infusion. On the other hand, approximately half (49% ± 11%) of the
CLL cells of patients in groups 1, 2, and 3 expressed the CD154
transgene, as assessed by flow cytometry (Figure 1A). Moreover, these
cells had an average mean fluorescence intensity ratio (MFIR) of
4.8 ± 1.6 when stained with a fluorochrome-labeled anti-CD154 mAb versus a fluorochrome-labeled, isotype-matched control mAb of irrelevant specificity. As expected, the expression of the CD154 transgene by Ad-CD154-infected CLL B cells resulted in increased and/or de novo expression of immune accessory molecules, for example, CD54, CD95, CD80, and CD86 (data not shown).
Immunologic response to Ad-CD154-transduced CLL cells Using reverse transcriptase (RT)-PCR, we detected expression of the CD154 transgene in the blood mononuclear cells of patients of groups 1 to 3 for at least 8 hours after the infusion of Ad-CD154-transduced leukemia cells (Figure 2). However, bystander leukemia cells that did not express the CD154 transgene had de novo or enhanced expression of CD80, CD86, CD54, and/or CD95 for several days after treatment (Figure 1B, Table 2, and data not shown). Such changes were not observed on the leukemia cells of patients in the pilot group, suggesting that a threshold number of infused Ad-CD154-transduced CLL B cells was required for this effect. Nevertheless, we did not otherwise discern a clear dose-response relationship between the number of infused Ad-CD154-transduced cells and the extent of the phenotypic changes on bystander cells of patients in groups 1, 2, and 3.
We examined whether there was a serum factor(s) induced by the infusion of Ad-CD154-CLL cells that could affect leukemia cell survival or expression of immune accessory molecules. For this, CLL cells obtained from patients before therapy were cultured for 24 hours in media supplemented with autologous serum collected before therapy and 8, 24, or 48 hours after the infusion of Ad-CD154-CLL cells. CLL cells cultured in media supplemented with 25% sera from any one of these time points had the same relative viability, as assessed by their ability to exclude propidium iodide (data not shown). Moreover, we did not observe any one of these sera to induce leukemia cells to increase their relative expression levels of CD80, CD86, or CD95 as assessed by flow cytometry (data not shown). High levels of Th1-type cytokines were detected in the plasma from
patients in groups 1 to 3 after infusion of Ad-CD154-CLL cells. Two of
the 3 patients in group 1 and all patients of groups 2 and 3 had high
plasma concentrations of IL-12 and IFN-
We also observed increases in absolute numbers of blood T cells
following the infusion of Ad-CD154-transduced cells. Such increases
followed the rise in cytokine levels by several days and lasted several
weeks. All 3 patients in group 1 had substantial increases
(206%-482%) in the absolute numbers of blood T cells by 1 to 4 weeks
after the infusion of Ad-CD154-transduced CLL cells (Table
4). Two of the 3 patients in group 2 had
significant increases (109% and 386%) in the numbers of T cells 1 to
3 weeks after treatment (Table 4). Finally, all 3 patients in group 3 had significant increases (97%-412%) in the absolute numbers of blood
T cells within 1 to 4 weeks after the infusion of Ad-CD154-transduced cells (Table 4). We observed increases in the absolute numbers of both
CD4+ and CD8+ T cells, commonly resulting in
absolute T-cell numbers of more than 4000 to 5000/µL of blood after
therapy (Table 4).
We used an ELISPOT assay to examine whether the increases in absolute
T-cell counts after treatment were associated with expansions in the
numbers of T cells that were reactive against autologous CLL B cells.
Blood T cells from patients before therapy and 4 weeks after treatment
were cultured for 48 hours with CD40-activated autologous CLL B cells.
The proportions of T cells induced to make IFN-
Patients' blood T cells collected several weeks after treatment also
had increased reactivity against autologous CLL B cells relative to
that of blood T cells collected before therapy. T cells were collected
6 months after treatment with Ad-CD154-CLL and examined for their
ability to mount MLRs against autologous CLL B cells. Such T cells
produced significantly greater amounts of IFN-
Clinical response to Ad-CD154-transduced CLL cells Because 97% or more of the blood lymphocytes of the patients in this study were CLL B cells (Tables 1 and 4), we monitored the absolute blood lymphocyte counts to follow the clinical response to treatment. Pilot study patients 001 and 002, who received autologous CLL cells of which only 4% or 8% expressed the CD154 transgene (Figure 2), did not have significant, sustained reductions in blood lymphocyte counts (Figure 5A). Although pilot group patient 002 did have a 30% or less reduction in blood lymphocyte count from pretreatment values, this was not sustained beyond 3 weeks after treatment. Moreover, 3 weeks after treatment, the blood lymphocyte counts of both patients in the pilot group persistently increased at rates similar to those observed before therapy (Figure 5A and data not shown), indicating that these patients continued to have progressive disease.
In contrast, the patients of groups 1 to 3 had significant reductions in blood lymphocyte counts (Figure 5A). The blood lymphocyte counts of 2 of the 3 patients in group 1 decreased by 68% and 87% from pretreatment values within 48 hours of infusion (Figure 5A). Moreover, these 2 patients had sustained reductions in lymphocyte counts of 66% and 65% within 2 to 4 weeks after treatment (Figure 5A). Similarly, all patients in group 2 experienced reductions in blood lymphocyte counts of more than 30% within 3 days and 54% to 66% within 1 to 2 weeks after receiving the infusion of transduced cells (Figure 5A). Furthermore, all patients in group 3 had reductions in blood lymphocyte counts of 66% to 83% within 2 to 3 days and sustained reductions of 49% to 55% within 2 to 4 weeks after the infusion of transduced cells. All patients of groups 1 and 3 had blood lymphocyte counts after therapy that were below that of pretreatment values. Moreover, in contrast to the patients in the pilot group, the patients in groups 1 to 3 did not show continued progressive increases in their blood lymphocyte counts for 3 months or more after treatment (Figure 5A and data not shown).The patients of groups 1 to 3 also experienced reductions in lymph node size 1 to 4 weeks after receiving a single infusion of autologous Ad-CD154-CLL cells (Figure 5B). All 3 patients in group 1 experienced a 64% to 90% reduction in lymph node size 2 to 4 weeks after therapy. Furthermore, all patients in group 2 experienced a 33% to 85% reduction, and all 3 patients in group 3 experienced 75% to 78% reductions in lymph node size within 1 to 4 weeks following therapy. In most cases, the reductions in lymph node size were sustained for 3 months or more after treatment (Figure 5B and data not shown). Safety The treatment was well tolerated with no immediate adverse reactions associated with infusion of Ad-CD154-transduced cells. The 2 patients in the pilot group did not have a discernible clinical response to the infusion. Patients in groups 1 to 3, however, generally developed flulike symptoms (eg, fever, fatigue, arthralgia, myalgia, nausea, and anorexia) by 12 hours after the infusion. Less common adverse reactions included headache, edema, dehydration, and diarrhea (data not shown). After treatment, a few patients developed mild, transient elevations in serum transaminase levels. Some patients had prolongation of prothrombin time, hypoalbuminemia, and/or a 40% or less reduction in platelet counts after treatment (data not shown).The coagulation abnormalities were not due to disseminated intravascular coagulation, in that no patients developed microangiopathic changes in their circulating red cells or had elevations in the plasma levels of fibrin-split products (data not shown). Rather, the prolongation in prothrombin time could be corrected by a 1:1 mix with control plasma and correlated with an acute, transient reduction in the level of factor VII (data not shown). Moreover, the decreases in platelet counts did not correlate with the administered dose and were transient, lasting less than 1 week. Both clinical and laboratory abnormalities were transient, were not dose limiting, and resolved within a few days after treatment. None of the treated patients developed immune thrombocytopenia, autoimmune hemolytic anemia, or a positive direct antiglobulin test. We did not identify a dose-limiting toxicity or a maximum tolerated dose in this study.
Hematologic malignancies are good candidate diseases for gene therapy in that large quantities of viable neoplastic cells can be readily harvested as single-cell suspensions for gene transfer ex vivo. Optimal methods for gene transfer and direct measurement of transgene expression can be achieved in vitro, allowing for controlled dose-escalation studies in which increasing numbers of transduced, autologous cells can be delivered back to the patient. Nevertheless, to date there have not been any published reports on clinical trials examining the use of gene transfer for the treatment of such diseases. We developed an immune gene therapy protocol for patients with CLL
based on in vitro studies demonstrating that CLL cells transduced with
Ad-CD154 became highly proficient at antigen presentation and could
induce autologous T-cell responses leading to the generation of
CLL-specific CTL.18 In these in vitro studies, CLL cells transduced with an adenovirus vector encoding an irrelevant protein ( Following infusion of autologous Ad-CD154-transduced cells, we observed phenotypic changes on bystander leukemia cells in vivo (Figure 1B and Table 2). This effect could not be mimicked by culturing the CLL cells in autologous sera obtained at various times after treatment, but rather resembled the bystander effect observed with Ad-CD154-transduced CLL cells on noninfected CLL B cells in vitro.18 This is despite the fact that the number of resident nontransduced leukemia greatly outnumbered the infused Ad-CD154-CLL B cells by estimated ratios of over 10 000:1. Such phenotypic changes were not restricted to only a subset of leukemia cells but were observed for the entire leukemia cell population. Because cytokines apparently could not effect such phenotypic changes in vitro, it is plausible that bystander leukemia cells are induced to express these surface antigens in secondary lymphoid compartments where there is a greater chance for cognate intercellular interactions to occur. If so, then the induced expression on the bystander leukemia cells seen in this study argues for a dynamic recirculation of leukemia cells between the blood and tissue compartments after treatment. We also noted high plasma levels of Th1-type cytokines soon after
the infusion of Ad-CD154-CLL cells. Indeed, significant levels of IL-12
and IFN- In addition, we also noted expansion of both CD4+ and
CD8+ T cells in the blood of patients 1 to 3 weeks after
therapy. ELISPOT and autologous MLR studies indicated that the infusion
of Ad-CD154-CLL cells enhanced the numbers of T cells that produce
IFN- The acute decreases in leukemia cell counts within 48 hours were unexpected. One possible explanation for the acute fall in circulating leukemia cell numbers includes leukemia cell homing or margination. However, none of the treated patients experienced a decrease in absolute lymphocyte count with signs of tissue infiltration or increases in lymph node size or vice versa. Instead, durable decreases in absolute leukemia cell counts were associated with reductions in lymph node size (Figure 5). As such, the infusion of autologous Ad-CD154 cells apparently resulted in an overall reduction in total tumor burden, even in patients with high leukemia cell counts and advanced disease. We reason that the acute decreases in leukemia cell counts most likely are due to leukemic cell apoptosis, possibly secondary to either alterations in leukemia microenvironment and/or the activity of an innate immune response to the infused cells. In any case, the initial rapid kinetics of the fall in leukemia cell counts indicates that this effect is not mediated by a primary leukemia-specific T-cell response. However, it is still possible that the rapid kinetics of leukemia cell clearance is secondary to the effects of a "recall" secondary immune response to the modified leukemia cells. Both the ELISPOT data (Figure 3) and the MLR data (Figure 4) indicate that there are leukemia-reactive T cells present in patients with this disease even before the infusion of the Ad-CD154-transduced cells. Moreover, the ability to induce leukemia-specific CTL after a few days in vitro is consistent with the notion that patients already are primed for a CTL response against target cells that express leukemia-associated antigens.18 This implies that activation of such a recall immune response can occur within the time period when the early reductions in leukemia cells counts are observed, thereby potentially contributing to leukemia cell clearance noted within the first few days after treatment. However, further studies are required to test this hypothesis. Although there did not appear to be clear dose-response
relationships between the patients of groups 1 to 3, there may be a
threshold below which there is no discernible immunologic or clinical
activity. Even though patients in group 1 received 10-fold fewer cells
than those in group 3, patients in both groups had sustained reductions
in blood lymphocyte counts and did not show signs of disease
progression after therapy. On the other hand, the patients in the pilot
group, who received cells expressing low levels of the CD154 transgene,
did not experience a significant decline in blood lymphocyte counts or
change in the disease progression after treatment. Furthermore, the
peak plasma levels of cytokines, such as IL-12 or IFN- Patients with CLL are at high risk for spontaneously developing autoimmune hemolytic anemia or immune thrombocytopenic purpura.23-25 One model proposes that aberrant expression of CD154 may be responsible for such autoimmunity in patients with CLL.26 An alternative model proposes that such autoimmunity may reflect immune dysregulation caused by an acquired deficiency of CD154 activity.27 In this regard, it is noteworthy that patients with congenital lack of CD154 also are at increased risk for developing autoimmune hemolytic anemia and immune thrombocytopenic purpura despite having a profound immune deficiency.28 In any case, none of the treated patients developed any signs of such autoimmunity despite receiving relatively large numbers of cells expressing CD154. Actually, the infusion of autologous Ad-CD154-CLL cells expressing high
levels of the CD154 transgene was well tolerated. Some patients
experienced laboratory abnormalities, such as elevation in hepatic
transaminase levels, reduction in platelet counts, or prolongation of
prothrombin times. These abnormalities were grade 2 or less in
magnitude and of limited duration. The clinical adverse reactions
primarily consisted of flulike symptoms that developed 6 to 8 hours
after infusion and lasted a few days. These clinical adverse reactions
most likely were secondary to the release of endogenous cytokines, such
as IFN- Because significant increases in T-cell numbers were observed following a single dose of Ad-CD154-transduced cells, a greater and more durable response is hypothesized to occur with repeat dosing. As discussed, pretreatment T-cell counts may have an impact on the initial response to treatment. Even patient 005, who had been pretreated with multiple cycles of chemotherapy and had very low pretreatment T-cell numbers, experienced an increase in the absolute numbers of CD4+ T cells after treatment (Table 4). Conceivably, subsequent infusions of autologous Ad-CD154-CLL cells when the T-cell counts are increased could induce more significant immunologic and clinical effects. As such, dosing schedule, rather than amount of cells per dose, may prove to be the critical factor for developing this into an effective and potentially curative gene therapy strategy for patients with CLL.
Submitted March 16, 2000; accepted June 20, 2000.
Supported in part by National Institutes of Health grant M01 RR00827, PO1 CA81534, R37 CA49870 (T.J.K.), and the California Division-American Cancer Society, Fellowship #4-22-98 (W.G.W).
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.
Reprints: Thomas J. Kipps, Division of Hematology and Oncology, Department of Medicine, University of California-San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0663; e-mail: tkipps{at}ucsd.edu.
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W. G. Wierda Current and Investigational Therapies for Patients with CLL Hematology, January 1, 2006; 2006(1): 285 - 294. [Abstract] [Full Text] [PDF]
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E. Biagi, R. Rousseau, E. Yvon, M. Schwartz, G. Dotti, A. Foster, D. Havlik-Cooper, B. Grilley, A. Gee, K. Baker, et al. Responses to Human CD40 Ligand/Human Interleukin-2 Autologous Cell Vaccine in Patients with B-Cell Chronic Lymphocytic Leukemia Clin. Cancer Res., October 1, 2005; 11(19): 6916 - 6923. [Abstract] [Full Text] [PDF]
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A. P. Kater, F. Dicker, M. Mangiola, K. Welsh, R. Houghten, J. Ostresh, A. Nefzi, J. C. Reed, C. Pinilla, and T. J. Kipps Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis Blood, September 1, 2005; 106(5): 1742 - 1748. [Abstract] [Full Text] [PDF]
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F. Dicker, A. P. Kater, T. Fukuda, and T. J. Kipps Fas-ligand (CD178) and TRAIL synergistically induce apoptosis of CD40-activated chronic lymphocytic leukemia B cells Blood, April 15, 2005; 105(8): 3193 - 3198. [Abstract] [Full Text] [PDF]
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E. Biagi, G. Dotti, E. Yvon, E. Lee, M. Pule, S. Vigouroux, S. Gottschalk, U. Popat, R. Rousseau, and M. Brenner Molecular transfer of CD40 and OX40 ligands to leukemic human B cells induces expansion of autologous tumor-reactive cytotoxic T lymphocytes Blood, March 15, 2005; 105(6): 2436 - 2442. [Abstract] [Full Text] [PDF]
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D. Watson, G. Y. Zhang, M. Sartor, and S. I. Alexander "Pruning" of Alloreactive CD4+ T Cells Using 5- (and 6-)Carboxyfluorescein Diacetate Succinimidyl Ester Prolongs Skin Allograft Survival J. Immunol., December 1, 2004; 173(11): 6574 - 6582. [Abstract] [Full Text] [PDF]
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A. Younes and M. E. Kadin Emerging Applications of the Tumor Necrosis Factor Family of Ligands and Receptors in Cancer Therapy J. Clin. Oncol., September 15, 2003; 21(18): 3526 - 3534. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
80°C. Mononuclear cells were isolated
via Ficoll-Hypaque density gradient centrifugation on Histopaque 1077 (Sigma Chemical, St Louis, MO). Following isolation, the cells were
washed and suspended in RPMI-1640. Autologous T cells were isolated
using anti-CD4 and anti-CD8-conjugated magnetic beads where indicated
(Dynal, Lake Success, NY). For this, the mononuclear cells
(1 × 108) were incubated with magnetic beads (200 µL/each) for 1 hour at 4°C. The estimated number of Dynalbeads per
T cell was 4 to 10. The magnetic bead-bound CD4+ and
CD8+ cells were washed and eluted by DETACHaBEAD (100 µL)
for 1 hour at room temperature. Over 95% of the isolated cells
expressed CD3 as assessed by flow cytometry (data not shown).
-galactosidase) were unable to induce autologous T cells, or even
allogeneic T cells, to proliferate or to secrete cytokines in an MLR.
We reasoned that autologous CLL cells transduced with Ad-CD154, but not
with a control adenovirus vector, could induce host antileukemia immune
responses in vivo. This formed the basis for the immune gene therapy
protocol described in this report.
an accumulative disease of immunologically incompetent lymphocytes.
Blood.
1967;29:566-584