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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-07-1989.
TRANSPLANTATION
From the Divisions of Pediatric Hematology/Oncology,
Molecular Medicine and Hematology and Bone Marrow
Transplant, and Department of Radioimmunotherapy, Beckman
Research Institute and City of Hope National Medical Center, Duarte,
CA; and Fred Hutchinson Cancer Research Center, Division of Clinical
Research, Seattle, WA.
Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after
allogeneic hematopoietic stem cell transplantation (HSCT) commonly
results from the failure of a graft-versus-leukemia (GVL) effect to
eradicate minimal residual disease. Augmenting the GVL effect by the
adoptive transfer of donor-derived B-ALL-specific T-cell clones is a
conceptually attractive strategy to decrease relapse rates without
exacerbating graft-versus-host disease (GVHD). Toward this end, we
investigated whether a genetic engineering approach could render
CD8+ cytotoxic T lymphocytes (CTLs) specific for tumor
cells that express the B-cell lineage cell surface molecule CD19. This
was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a
CD3- The ability of allogeneic hematopoietic stem cell
transplantation (HSCT) to eradicate hematologic neoplasia is dependent
not only on the cytotoxic effects of high-dose chemotherapy and
chemoradiation therapy, but the subsequent immune-mediated destruction
of neoplastic cells via effector cells derived from the donor
hematopoietic stem cell (HSC) graft.1 The potency of the
graft-versus-leukemia (GVL) effect varies widely depending on the type
of leukemia. For B-lineage acute lymphoblastic leukemia (B-ALL), the
GVL effect is modest and consequently disease relapse after
transplantation is a major contributor to treatment failure. Although
intensifying cytotoxic conditioning regimens has diminished relapse
rates, the benefit of this approach to improve disease-free survival is
frequently mitigated by the increased regimen-related
toxicities.2-4
Alternately, the problem of B-ALL posttransplantation relapse can be
approached by therapeutic strategies to augment the GVL effect. The
adoptive transfer of donor leukocytes following HSC engraftment can
induce durable remissions particularly in patients with chronic
myelogenous leukemia (CML). However, donor lymphocyte infusions (DLIs)
achieve remission rates of less than 10% in patients with B-ALL and
are associated with a high incidence and severity of graft-versus-host
disease (GVHD) morbidity and mortality.5,6 Although the
genetic modification of donor lymphocytes to introduce suicide genes
may improve the safety profile of DLIs by rendering transferred
lymphocytes susceptible to in vivo ablation, the low potency and high
association with GVHD continue to be major obstacles to achieving
clinically robust anti-B-ALL GVL augmentation by this
approach.7-9
Adoptive transfer of donor-derived T cells having antigen specificity
restricted to the leukemic clone itself or to target antigens
selectively expressed by host hematopoietic lineage cells has the
potential to selectively augment GVL without exacerbating GVHD. Minor
histocompatibility antigens (mHags) encoded by polymorphic genes
selectively expressed in recipient leukemic cells/hematopoietic-derived cells can serve as target antigens for donor T cells and mediate selective tumor eradication.10-15 Significant challenges
face the widespread implementation of adoptive therapy with donor
mHag-specific T cells; these include the molecular identification of a
panel of mHags with restricted hematopoietic expression, the
delineation of immunogenic epitopes for a large number of HLA alleles,
and the technologies for reliably isolating T cells with desired
specificities from donors.16,17 Additionally, tumor escape
mechanisms such as antigen-loss variants, the immune-mediated selection
of leukemic clones that have down-regulated restricting HLA alleles, or
critical adhesion/costimulatory molecules will need to be addressed,
particularly for B-ALL.18
The genetic modification of T cells to introduce antigen receptors that
are capable of recognizing leukemic cells is a strategy that can
overcome the requirement of isolating leukemia/mHag-specific T cells
endogenous to the donor. Moreover, chimeric immunoreceptors that use
antibody-derived single-chain variable domains (scFvs) bind to native
cell surface epitopes and bypass the requirement for antigen processing
and presentation by HLA molecules and are therefore
universal.19,20 We have constructed a chimeric
immunoreceptor having a CD19-specific scFv extracellular targeting
domain that activates T cells via its cytoplasmic CD3- Here we demonstrate that primary human CD8+ cytotoxic T
lymphocyte (CTL) clones expressing our CD19-specific chimeric
immunoreceptor specifically recognize and lyse CD19+
leukemia/lymphoma cells and primary B-ALL blasts and are activated for
chimeric immunoreceptor-regulated cytokine production and proliferation. Additionally, we find that the high efficiency of tumor
cell killing/CTL activation achieved via our chimeric immunoreceptor is
independent of disparities in the expression levels of adhesion
molecules on leukemic cells that participate in the formation of a
productive immunologic synapse, an escape mechanism relevant to
B-ALL.
Expression plasmid
Cells
Genetic modification
Flow cytometry The cell surface phenotype of the genetically modified T cells and tumor lines was determined by staining with the following: fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated mAbs specific for T-cell receptor /
(TCR), CD2, CD3 , CD4, CD8, CD11a, CD16, CD18, CD28, CD54,
CD57, and CD58 (BD Biosciences, San Jose, CA). Surface expression of
the CD19-specific immunoreceptor was detected using the
F(ab')2 fragment of FITC-conjugated goat antibody specific
for human IgG Fc fragment (Immunotech, Marseilles, France). Briefly,
105 to 106 washed cells were resuspended in 100 µL Hanks balanced salt solution (HBSS) supplemented with 2%
FCS, 0.2 mg/mL NaN3 staining buffer, and 0.5 to 2 µL of
the stock antibody preparation. Following a 60-minute incubation on
ice, the cells were washed twice and resuspended in staining buffer
containing propidium iodide (PI) at 1 µg/mL. Dead cells were excluded
from analysis on uptake of PI. Intracellular staining with
PE-conjugated mAb specific for perforin (BD Biosciences) was undertaken
on T cells that had been fixed and permeabolized using the Pharmingen
(BD Biosciences) system. Cells were analyzed on a FACSCalibur cytometer
(BD Immunocytometry Systems, San Jose, CA). CellQuest software (BD
Immunocytometry Systems) was used to calculate the percentage of cells
and mean fluorescent intensity (MFI) within a given region of a histogram.
Western blot Expression of the CD19R was determined by Western blot using a mAb specific for CD3- to detect the chimeric immunoreceptor. Whole
cell lysates of transfected T-cell clones were prepared by lysis of
106 washed cells in 1 mL RIPA buffer (phosphate-buffered
saline [PBS], 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate [SDS]) containing 1 tablet/10 mL Complete Protease
Inhibitor Cocktail (Boehringer Mannheim, Penzberg, Federal Republic of
Germany). After a 60-minute incubation on ice, aliquots of centrifuged
whole cell lysate supernatant were harvested and boiled in an equal volume of loading buffer under reducing conditions and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on precast 12%
acrylamide gels (Bio-Rad Laboratories, Hercules, CA). Following transfer to nitrocellulose, membranes were blocked for 2 hours in
Blotto solution containing 0.07 g/mL nonfat dried milk. Membranes were
washed in T-TBS (0.05% Tween 20 in Tris
[tris(hydroxymethyl)aminomethane]-buffered saline, pH 8.0) and then
incubated with primary mouse anti-human CD3- mAb 8D3 (Pharmingen,
San Diego, CA) at a concentration of 1 µg/mL for 2 hours. Prior to
developing, the membranes were washed 4 times in T-TBS and then
incubated for 1 hour with a 1:500 dilution of alkaline
phosphatase-conjugated goat antibody specific for murine IgG. After
rinsing in T-TBS the membranes were developed with 30 mL AKP solution
(Promega, Madison, WI) per the manufacturer's instructions.
Chromium release assay The cytolytic activity of genetically modified CD8+ T-cell clones was determined in a 4-hour chromium release assay (CRA) using 51Cr-labeled RS4, Daudi, JM-1, SupB-15, and erythroleukemia K562 target cells. The T-cell effectors were harvested 12 to 14 days following stimulation with OKT3, washed, and plated in V-bottom microtiter plates (Costar, Cambridge, MA) at 37°C for 4 hours in triplicate at 2.5 × 105, 1.25 × 105, 0.25 × 105, and 0.05 × 105/well with 5 × 103 target cells. After centrifugation and incubation, 100-µL aliquots of cell-free supernatant were harvested and counted and the percent specific cytolysis was calculated from the release of 51Cr as follows:
Cytokine production Duplicate wells containing 106 cells of a genetically modified CD8+ T-cell clone were coincubated with 106 stimulator cells irradiated to 8000 cGy in 2 mL culture media for 72 hours with rhIL-2 at 5 U/mL. Cell-free supernatants were harvested and assayed for cytokine content by enzyme-linked immunosorbent assay (ELISA; R & D Systems, Minneapolis, MN) and the concentration was extrapolated from a standard curve.T-cell proliferation Cells (105) of a CD19-specific CD8+ T-cell clone were cocultured with 2 × 106 irradiated PBMCs, with and without a panel of 105 irradiated stimulator cells. Parallel culture conditions were established without the addition of genetically modified T cells. T cells were also plated without PBMCs and stimulator cells. Then, rhIL-2 at 5 U/mL was added every 48 hours. The viable cells, based on exclusion with trypan blue, were enumerated at day 7 and day 14. On day 7 of the culture, 2 × 106 irradiated PBMCs were re-added with and without the irradiated stimulator cells at a 1:1 ratio with the T cells. The assay was repeated 3 times. Fold expansion was calculated by dividing the average cell counts by the input T-cell number of 105. The proliferative activity was also measured by means of a [3H]thymidine incorporation assay. T cells (105) were cocultured with 106 irradiated PBMCs with and without irradiated 105 Daudi and LCL stimulator cells. Then, rhIL-2 at 5 U/mL was added at the beginning of the assay and again at 48 hours. On the fourth day of culture [3H]thymidine (ICN, Costa Mesa, CA) was added at a concentration of 50 µCi/mL (7.4 MBq). The following day the incorporation of [3H]thymidine into T cells was measured by seeding the culture wells into replicates of 6 wells on 96-well plates and harvesting the cells on glass fiber filters with a PHD Harvester (Brandel, Gaithersburg, MD), and then radioactivity was measured with a liquid scintillation LS 6500 counter (Beckman Coulter, Fullerton, CA). Data are reported as an average ± SD.
Genetically modified T-cell clones express the CD19-specific chimeric immunoreceptor To generate CD19-specific T cells that coexpress CD19R and HyTK genes, PBMCs were activated with OKT3 mAb specific for CD3 and the naked DNA linearized plasmid CD19R/HyTk-pMG was introduced by electrotransfer. Four weeks after electroporation, an outgrowth of cells in the presence of cytocidal concentrations of hygromycin was observed. Culture systems were developed to retrieve clones of stable transfectants by plating T cells in limiting dilution. This procedure has yielded drug-resistant clones in multiple separate electroporations of T cells and these clones can be expanded to 1010 in number over approximately 10 weeks with retention of both transgene expression and redirected CTL effector function. Although these culture conditions tend to favor the emergence of CD8+ CTL clones whose growth is supported by the addition of rhIL-2, CD4+ CTL clones can be identified. Genetically modified hygromycin-resistant CD8+ T-cell clones were evaluated for expression of the chimeric immunoreceptor by Western blot of reduced whole cell lysates probed using a mAb specific for CD3- chain. This technique revealed that the genetically modified T cells express the introduced 66-kDa chimeric chain in
addition to the 21-kDa endogenous chain (Figure
2A). An additional band between 21 and 66 kDa was routinely observed in both unmodified and genetically modified
T cells and is of uncertain significance. Flow cytometry using mAbs
specific for CD8, CD4, TCR, and F(ab')2 fragments of
goat polyclonal antibody specific for human Fc were used to validate
that the majority of genetically modified hygromycin-resistant T-cell
clones from healthy donors uniformly express
CD8+CD4 TCR+Fc+
on the surface (Figure 2B). However, as is typical for CD8+
T cells expanded ex vivo using the described techniques, these differentiated effector cells failed to express CD28, rendering these
CTLs unable to receive costimulatory signals from the binding of CD28
to B7 molecules on tumor cells.
CD8+ T cells induce GVL cytotoxic effects primarily through perforin-mediated cytotoxicity by releasing granule contents after target cell recognition.41 Therefore, we investigated the perforin content in the genetically modified CTLs using flow cytometry and found that the CD8+ CD19-specific T cells uniformly express the cytotoxic effector protein perforin (Figure 2B). Because human natural killer (NK) cells, expressing CD57 or CD16 or both, can also exhibit cytotoxicity, we used the lack of surface expression of these markers to distinguish the genetically modified T-cell clones from NK/T cells. CD19+ tumor lines express variable levels of T-cell adhesion molecules A panel of CD19+ B-ALL and lymphoma lines was assembled to investigate the role of adhesion molecules in the effector function of CD19-specific T cells. The adhesion molecules were selected based on an ability to form stable conjugates between T cells and target cells, thereby contributing to the assembly of the immune synapse. For example, the binding of intercellular adhesion molecule-1 (ICAM-1, CD54) with lymphocyte function-associated antigen-1 (LFA-1, CD11a/18) and CD2 with LFA-3 (CD58) can facilitate T-cell coactivation on docking of the TCR with cognate antigen.42-44 The relative cell surface expression of these adhesion molecules on the tumor lines was demonstrated by flow cytometry using a panel of adhesion molecule-specific mAbs. Even though CD19-specific CD8+ T cells expressed relatively high levels all of the accessory molecules tested, the pattern of expression of these molecules varied on the CD19+ tumor lines, both in percentage of cells expressing adhesion molecules and the density of adhesion molecules detected on the surface (Figure 3). The flow cytometry data revealed that the RS4 and SUP-B15 cells expressed the lowest levels of CD11a and CD18, whereas Daudi and JM1 cells expressed the lowest levels of CD58 and the expression of CD54 was reduced on SUP-B15 cells. Furthermore, there was variation between the tumor lines in the density of adhesion molecule expression as measured by MFI. For example, there was approximately a 10-fold range in the MFI associated with CD54 and CD58 on tumor lines that uniformly express these determinants. However, whereas the leukemia and lymphoma cell lines, RS4, Sup-B15, Daudi, and JM-1 varied in the relative expression and intensity of anti-ICAM-1, LFA-1, and LFA-3 staining, the EBV-transformed LCLs uniformly expressed the highest levels of these adhesion molecules.
CD19+ tumor lines are lysed equivalently by CD19-specific T cells We investigated whether the altered expression of adhesion molecules on CD19+ tumor lines correlated with a susceptibility to be lysed by CD19-specific CD8+ T cells. These B-ALL and lymphoma lines expressed approximately equivalent levels of CD19 determinant; thus, differences in lysis could not be explained by variation of antigen expression (Figure 3). The lines were used as targets in a 4-hour CRA and the analysis revealed that the ability of a genetically modified T-cell clone to lyse the CD19+ lymphoma and leukemia lines was independent of the relative expression of LFA-1, LFA-3, and ICAM-1. Furthermore, both CD8+ and CD4+ genetically modified CTL clones efficiently killed HLA-mismatched human CD19+ leukemia and lymphoma cells because maximal lysis was achieved with an effector-target ratio of 5:1 (Figure 4). The genetically modified T cells preserve their cytolytic activity after extensive in vitro culture because the results from the 4-hour CRA presented are from a CD19-specific T-cell clone that has been cultured in vitro for approximately 100 days. The specificity of the genetically modified T cells for CD19 was demonstrated by the relative lack of lysis of the K562 CD19 cell line (Figure 4) and
lack of lysis of the CD19Be-2 neuroblastoma line (Figure
7). In addition, CD8+ T cells genetically modified to be
CD20 specific (M.C.J., L.J.N.C., A. M. Wu, S.J.F., A.R.,
manuscript submitted) fail to lyse the CD20CD19+ JM1 leukemia line, but can lyse
CD20+CD19+ Daudi cells, indicating that the
specificity of activation is mediated via the specificity of the
chimeric immunoreceptor's scFv (Figure 4 insert right).
CD19-specific T cells produce cytokines in response to stimulation with CD19+ tumor cells The ability of T cells to modulate cytokine secretion in response to antigenic stimulation is important for an effective immune response.45,46 To test whether the genetically modified T-cell clones are activated via the chimeric immunoreceptor for regulated CD19-specific cytokine secretion in response to human CD19+ leukemia and lymphoma cells, the CD19-specific T-cell clones were cocultured with the panel of tumor cells. Conditioned supernatants from these cultures were tested for production of the cytokines GM-CSF, tumor necrosis factor (TNF-), and interferon
 (IFN-). The results for a CD8+ CD19-specific
cytolytic T-cell clone are shown in Figure
5. The genetically modified T-cell clone
produced cytokines in response to each of the CD19+
leukemia and lymphoma stimulator cells, but not by a CD19
stimulator cell (K562), nor by media alone. The T-cell production of
IFN- in response to each CD19+ tumor line was
approximately equivalent, whereas RS4 and SUP-B15 cells were able to
stimulate the highest levels of TNF-, and Daudi cells and LCLs
stimulated the highest levels of GM-CSF. Thus, the CD19-specific T
cells could be activated to secrete Tc1 cytokines despite
alterations in the expression of adhesion molecules on the tumor
cells.
CD19-specific T cells proliferate in response CD19+ tumor lines A proliferative response of CD19-specific CTLs to CD19+ tumor targets indicates that the chimeric immunoreceptor is competent to deliver a stimulatory response for growth and establishes that the docking of genetically modified T cells with CD19+ tumor cells does not lead to T-cell degradation. Figure 6 demonstrates that a CD8+ CD19-specific T-cell clone could exhibit up to an 8-fold expansion in numbers over a 2-week period when cocultured with CD19+ leukemia and lymphoma stimulators. The proliferation of genetically modified T cells in response to the leukemia SUP-B15 and lymphoma Daudi lines was superior to using LCLs as stimulators, despite the decreased expression of adhesion molecules relative to LCLs. Furthermore, the proliferation of the genetically modified T-cell clones in response to Daudi stimulators was similar to the cell growth achieved using OKT3, PBMCs, and LCLs to activate the CTL culture conditions that are similar to those used for the in vitro expansion of
T cells. The proliferation of CD19-specific T cells was dependent on
the expression of CD19 on tumor cells because tissue culture conditions
lacking CD19+ tumor cells resulted in only minimal T-cell
expansion. Similar results were obtained using an assay that measured
the incorporation of [3H]thymidine by proliferating T
cells (Figure 6 insert). Each of the culture conditions included
low-dose rhIL-2 added at 5 U/mL; however, the use of this cytokine
alone was insufficient to sustain the survival of the CD19-specific
CD8+ T cells, as demonstrated both by the failure of the T
cells to proliferate within 14 days when cultured with rhIL-2 in the
absence of CD19+ tumor cells, and by the correspondingly
near-background incorporation of [3H]thymidine by the T
cells. The addition of PBMCs, containing normal CD19+ B
cells, along with low-dose rhIL-2, could maintain the survival of the
genetically modified T cells, but this condition resulted in only a
2-fold expansion and was associated with background levels of
[3H]thymidine incorporation.
CD19-specific T cells recognize CD19+ primary B-ALL cells The ability of CD19-specific T cells to recognize B-ALL and lymphoma lines suggested that these genetically modified T cells might also recognize primary B-ALL cells. Cryopreserved blasts were used as targets in a 4-hour CRA to test this hypothesis. Figure 7 demonstrates that a CD19-specific CTL clone could lyse CD19+ blasts almost to the same extent as the CD19+ JM1 leukemic line. A CTL clone genetically modified to express a chimeric immunoreceptor specific for a neuroblastoma-specific antigen failed to recognize the blasts, but did lyse the CD19Be-2 neuroblastoma line (Figure 7 insert).
These data indicate that the CD19-specific immunoreceptor is sufficient
to trigger killing of primary CD19+ leukemia
cells.
Our data demonstrate that CD19 can serve as a target for primary human CTL clones with specificity redirected via a CD19-specific chimeric immunoreceptor. CD19+ lymphoma and leukemic lines, as well as primary B-ALL cells, are efficiently killed by clonal populations of these engineered effector cells although the chimeric immunoreceptor also regulates CD19-specific cytokine secretion and proliferation. We observed that the efficiency of CTL activation for these effector functions by B-ALL and lymphoma cells was not impaired despite the low-level expression of ICAM-1, LFA-1, and LFA-3 accessory molecules on target cells. This result is likely attributable to the high affinity of the chimeric immunoreceptor's scFv for CD19 as a means to overcome the impaired avidity of the T-cell immunologic synapse with targets expressing low levels of ICAM-1 or LFA-1 or LFA-3. Down-regulation of these accessory molecules is common in B-ALL and is likely to be an immunologic escape mechanism used by leukemic cells to avoid recognition by T cells and NK cells.47-52 CD19 represents a ubiquitous target moiety for B-lineage leukemias and
lymphomas. Targeting CD19 with mAb-based therapeutics is the subject of
clinical investigation and has not, to date, revealed a high incidence
of selection for CD19 Because nonmalignant CD19+ B cells will be subject to recognition by redirected CTLs, the persistence of the adoptively transferred CD19-specific CTLs has the potential to exacerbate/prolong the B-cell immunodeficiency associated with allogeneic HSCT. However, the in vivo persistence of the genetically modified T cells can be limited by using CD19-specific CD8+ CTLs that are dependent on exogenous rhIL-2 for survival, or by coexpression of a suicide gene, such as HSV-TK. In patients who are predicted to relapse with B-ALL after allogeneic HSCT, the clinical sequelae of temporary B-cell lymphopenia may be an acceptable side effect of CD19-directed immunotherapy, especially because prolonged ablation of normal CD20+ B cells in patients receiving rituximab therapy does not appear to result in clinically significant complications attributable to depleted numbers of normal B cells.57 Although the safety of the patient in initial adoptive immunotherapy trials may be enhanced through the coexpression of a suicide gene along with the CD19-specific immunoreceptor in T-cell clones, the expression of the HyTK gene may limit the in vivo survival of the genetically modified T cells, as a result of an immune response directed against the transgenes. To reduce the immunogenicity of genetically modified T cells, our laboratory is exploring the use of flow cytometry operating in compliance with current good manufacturing practices (cGMPs) to sort on T cells displaying an introduced chimeric immunoreceptor that have not undergone hygromycin drug selection, humanizing the CD19-specific scFvs, and use of the human Fas-based suicide switch.58 Although cloned genetically modified CTLs represent a homogeneous
high-potency cell product for clinical experimentation that maximizes
safety due to dependency on exogenous rhIL-2, the optimal cell product
will have to account for the potential adverse impact of immunogenicity
of expressed transgenes, the lack of costimulatory ligands on B-ALL,
and deficits in homing of ex vivo propagated T cells to all tissue
compartments harboring leukemic blasts. Under development is the
further engineering of CTLs to express engineered costimulatory
receptors to enhance their function and prevent activation-induced cell
death,59 and the genetic modification of clones to express
selectin/chemokine receptor pairs for specified tissue
homing.60-62 Our naked DNA T-cell transfection procedure provides for ample flexibility to engineer polycistronic vectors and is
a cost-effective manufacturing practice to produce clinical grade
material. Finally, as Brenner et al have suggested, future studies will
also use T cells that are selected on the basis of the clone's
endogenous These preclinical data lay the groundwork for clinical trials to
determine the safety, feasibility, and efficacy of using donor-derived
CD19-specific CTL clones after allogeneic HSCT. Using cGMP-compliant
methodologies currently used in Food and Drug
Administration-authorized clinical trials at City of Hope National
Medical Center for isolating and expanding plasmid vector-modified CTL
clones, we will be initiating a clinical trial in which patients experiencing a molecular B-ALL relapse will be treated with escalating cell doses of donor-derived CD19-specific CTL clones selected on the
basis of expressing an endogenous
Submitted July 5, 2002; accepted September 27, 2002.
Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-07-1989.
Supported by CA30206, CA33572, Leukemia and Lymphoma Society, Altschul and Abe and Estelle Sanders foundations, Zagoria Foundation, Deutsche Forschungsgemeinschaft (To208/1-1) and the Cancer Research Institute.
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: Laurence J. N. Cooper, Division of Molecular Medicine Beckman Research Institute, Division of Pediatric Hematology/Oncology, City of Hope National Medical Center, 1500 E Duarte Rd, Duarte, CA 91010; e-mail: lcooper{at}coh.org.
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Abstract
Introduction
), SUP-B15 (
),
JM1 (
), RS4 (
), and CD19
