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ARTICLE doi:10.1016/j.ymthe.2004.09.006
IL-2/B7.1 (CD80) Fusagene Transduction ofAML Blasts by a Self-Inactivating Lentiviral VectorStimulates T Cell Responses in Vitro: a Strategy to
Generate Whole Cell Vaccines for AML
Lucas Chan,1,* Nicola Hardwick,1,* Dave Darling,1 Joanna Galea-Lauri,1
Joop Gaken,1 Steve Devereux,1 Mike Kemeny,2 Ghulam Mufti,1 Farzin Farzaneh,1,y
1Department of Hematological and Molecular Medicine and 2Department of Immunology, Guy’s, King’s, and St. Thomas’ School of Medicine,
University of London, London SE5 9NU, UK
*These authors contributed equally to this publication.
yTo whom correspondence and reprint requests should be addressed. Fax: (+44) 20 7773 3877. E-mail: farzin.farzaneh@kcl.ac.uk.
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Combined expression of costimulatory factors and proinflammatory cytokines stimulate effectiveimmune-mediated tumor rejection in a variety of murine tumor models. Specifically, syngeneic tumorcells genetically modified to express B7.1 (CD80) have been shown to induce rejection of previouslyestablished murine solid tumors, and transduction with IL-2 can further increase survival. However,poor rates of gene transfer and inefficient expression of multiple transgenes encoded by singlevectors have hampered the development of such autologous tumor cell vaccines for clinical trials inacute myeloid leukemia (AML) patients. Here we describe the development of a self-inactivatinglentiviral vector encoding B7.1 and IL-2 as a single fusion protein postsynthetically cleaved togenerate biologically active membrane-anchored B7.1 and secreted IL-2. This enables the efficienttransduction of both established and primary AML blasts, resulting in expression of the transgenes inup to 98% of the cells following a single round of infection at an m.o.i. of 10. The combinedexpression of IL-2 and B7.1 in AML blasts enables increased stimulation of both allogeneic andautologous T cells. The stimulated lymphocytes secrete greater levels of Th1 cytokines and showevidence of specificity, as indicated by their increased proliferation in the presence of autologousAML compared to remission bone marrow cells.
Key Words: HIV, lentivirus, AML, immunotherapy, B7.1, IL-2
INTRODUCTION
The prognosis for acute myeloid leukemia (AML) hasimproved considerably in the past 2 decades due toadvances in intensive chemotherapy and allogeneic bonemarrow transplantation (BMT). However, outcome forthe majority of AML patients remains bleak. The need toimprove prognosis in this disease has therefore led to thesearch for alternative therapies. This includes harnessingthe graft versus leukemia effect using reduced intensityconditioned allogeneic hematopoietic stem cell trans-plantation (for review, see [1]). However, to date this hasbeen of limited effectiveness.
In contrast to solid tumors, large numbers of viableAML blasts are obtainable from patients by leukapho-resis. Therefore this provides a readily available source ofcells for potential use as cancer vaccines. In addition,
AML blasts are of the same lineage as professionalantigen-presenting cells (APCs); hence, unlike manyother tumor cells, they express high levels of MHC-IIand adhesion molecules such as ICAM-1. Therefore AMLpresents a unique opportunity for immune-based thera-pies (for review see Galea-Lauri [2]). However, unlikeprofessional APCs, most AML blasts lack the costimula-tory molecule B7.1 (CD80) [3–6] and secrete immuno-suppressive factors [7]. A logical approach is therefore tomodify AML blasts to express B7.1 plus a stimulatorycytokine.
There is evidence in both solid tumor models [8,9] andleukemia [10–12] that B7.1 is an effective stimulator ofanti-tumor immune responses. Furthermore, evidence innonleukemic models reported by us [13,14] and others[15,16] shows that this can be enhanced by the combined
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use of IL-2. To date there are limited reports of the B7.1and IL-2 combination in leukemia [17] but a recent casestudy in a single AML patient is encouraging [18]. A Th1cytokine, such as IL-2, is a logical choice, as CTLresponses, rather than antibody responses, are consideredto offer the greatest promise for successful immunother-apy. The hyporesponsive state of T cell anergy is thoughtto aid tumor evasion of the immune response. Stimula-tion of anergic T cells with IL-2 results in proliferationand a complete reversal of the state [19]. In addition,systemic IL-2 therapy has been reported to have benefi-cial effects in AML patients but problems of toxicityprevent its widespread application [20].
Until recently, expression of transgenes in humanAML blasts has been limited to the use of retroviral [3,21]and adenoviral vectors [21], which are poorly effectivedue to the low proliferation of primary AML blasts andvariable coxsackie adenovirus receptor expression[22,23], respectively. In addition, the use of vectorsbased on herpes simplex virus and adeno-associatedvirus gives highly variable transduction efficiencies (ourown unpublished observations). This has hampereddevelopment of immune gene therapy for AML.
HIV-1-based lentiviral vectors, in contrast to classicalretroviral vectors, readily transduce mitotic as well as anumber of postmitotic targets, owing to the HIV matrixprotein as well as the accessory protein Vpr [24,25].While optimized attenuation of viral genes improves thesafety of the packaged lentiviral vector [26], the incor-poration of the HIV central polypurine tract (cppt) andtermination sequence (cts) improves nuclear import inotherwise nonpermissive targets [27,28]. In addition, thepresence of the woodchuck hepatitis virus posttranscrip-tional regulatory element (WPRE) improves intracellularmessenger RNA stability [29]. Enhanced lentiviral vector-
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derived transgene expression in hematopoietic cells hasbeen observed by the incorporation of these elementsindividually and in combination [30,31]. The absence ofany viral-encoded proteins warrants lentiviral vectors assuitable vehicles for immune gene therapy strategies.The effectiveness of lentiviral transduction on leukemiccells has been previously demonstrated by several groups[32–36], including the transfer of B7.1/GM-CSF incombination, which resulted in autologous T cellstimulation.
In our development of whole cell vaccines for AML,we show for the first time an efficient transductionprotocol to modify primary AML blasts to express B7.1and IL-2 as a single cistron from a myeloid efficientself-inactivating lentiviral backbone. Furthermore, weshow that primary AML blasts modified in this way arecapable of stimulating enhanced in vitro responses inallogeneic, chimeric, and autologous settings.
RESULTS
Improved Self-Inactivating (SIN) Lentiviral ExpressionVectorsThe lentiviral (LV) expression vectors were derivedfrom the self-inactivating vector backbone carrying adeletion in the HIV U3 promoter region of the 3V LTR,which results in its inactivation following reversetranscription in the target cells [37]. To achieveefficient transgene expression, we incorporated theHIV flap region encompassing the cppt and cts, aswell as the WPRE. To promote transgene expression,we used the U3 promoter sequence of the spleen focus-forming virus (SFFV) LTR, which is highly active inmyeloid lineage-committed cells [38]. The resultingvector, HRVSINctwSV, was used to express: (1) GFP
FIG. 1. Schematic representation of SIN
lentiviral vectors expressing GFP, B7.1, IL-2,
or B7.1 + IL-2. Common features of the
lentiviral backbone include 5V LTR, C—pack-
aging signal, RRE (Rev-responsive element),
flap—HIV central polypurine tract and termi-
nal sequence, SFFV (spleen focus-forming
virus) LTR U3 region, WPRE (woodchuck
hepatitis posttranscriptional regulatory ele-
ment), and the U3-deleted HIV 3V LTR, which
renders the vector self- inactivating.
HRVSINctwSVGFP (LV.GFP) expresses the
enhanced green fluorescence protein,
HRVSINctwSVB71 (LV.B7.1) expresses the
1074-bp B7.1 cDNA, HRVSINctwSVIL2
(LV.IL-2) expresses the 459-bp human IL-2,
and HRVSINctwSVIL2B71 (LV.IL-2/B7.1)
expresses the fusion IL-2/B7.1 separated by
a furin cleavage site (Arg.Gly.Arg.Arg). ER
transport of chimeric IL-2/B7.1 is mediated
by the IL-2 signaling peptide located at the
N-terminus.
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(LV.GFP), (2) B7.1 (LV.B7.1), (3) soluble IL-2 (LV.IL-2),and (4) IL-2 plus B7.1 as a chimeric monocistronictranscript (LV.IL-2/B7.1) with the leader sequencelocated at the N-terminus of IL-2 and separated fromthe mature B7.1 by a furin endoprotease cleavage site—Arg.Gly.Arg.Arg. The fusion protein encoded by LV.IL-2/B7.1 is postsynthetically cleaved in the Golgi togenerate the biologically active constituents [39]. SeeFig. 1 for vector details.
In vitro Characteristics of Primary Myeloid LeukemiaCellsWe included in the study only samples that thrived inculture, as defined by at least 80% viable cells after 1week of culture, as determined by flow cytometry basedon forward and side scatter characteristics (data notshown). We examined samples for FAB subtypes andkaryotypes (Fig. 2) and for the expression of thefollowing cell surface markers: CD34, CD14, CD19,CD3, MHC-I, MHC-II, CD80, and CD86 (data notshown). We included only samples with a high levelof purity, as defined by at least 70% CD34-positive cells.All AML blasts expressed high levels of MHC-I, MHC-II,and CD86 (data not shown) but undetectable levels ofCD80 (Fig. 4A).
Lentiviral Vectors Mediate Efficient Transduction ofboth Established and Primary Myeloid Leukemia CellsWe tested the efficiency of lentiviral vectors first onleukemic cell lines to determine optimal m.o.i. Weinfected U937, K562, and NB4 cells with LV.GFP and
FIG. 2. Infection of U937, NB4, and
K562 leukemic cell lines by SIN lentiviral
vectors. FACS profiles of leukemia cells
infected with LV.GFP. (A) U937, K562,
and NB4 inoculated at an m.o.i. of 0.2
(gray lines, clear background) and 2
(black lines, clear background), analyzed
8 days after infection. (B) Primary AML
sample from Patient 1 infected at an
m.o.i. of 3 and analyzed 4 days after
infection.
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analyzed them by FACS 8 days later to exclude thepossibility of pseudotransduction [40]. We observedsimilar efficiencies in gene transduction for all threecell lines, providing between 20 and 99% modificationat m.o.i. of 0.2 and 2, respectively (Fig. 2A). GFPtransgene expression was remarkably high, resulting inbetween 3 and 4 log increases in fluorescence. When weused the same virus preparation to infect primary AMLblasts, we observed an equally efficient gene trans-duction at an m.o.i. of 3 (Fig. 2B shows Patient 1 as arepresentative sample). These data demonstrate thesuitability of the constructed lentivirus for efficienttransduction in both established and primary myeloidleukemia cells at relatively low m.o.i. with a singleround of infection.
Modification of AML Blasts to Express B7.1 and IL-2Alone or in Combination by Lentiviral VectorsWe evaluated LV.B7.1, LV.IL-2, and LV.IL-2/B7.1 oneight primary AML samples and on U937 and NB4 celllines (Fig. 3A). Lentiviral transduction had no signifi-cant effect on viability of either primary or establishedcells, as represented by the Viability Index. Similar toLV.GFP transduction, the established U937 and NB4cell lines were readily transduced by both LV.B7.1 andLV.IL-2/B7.1 at an m.o.i. of 10, producing greater than95% transduced cells as determined by the surfacedetection of B7.1. Transduction of primary AML blastsby LV.B7.1 and LV.IL-2/B7.1 was, however, morevariable, with B7.1 modification ranging from 60 to98% for LV.B7.1 and 41 to 98% for LV.IL-2/B7.1. This
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yFIG. 3. Transduction of established and primary leukemic samples by lentiviral vectors expressing B7.1 (LV.B7.1), IL-2 (LV.IL-2), and IL-2/B7.1 (LV.IL-2/B7.1). (A)
Eight primary AML samples (Pats. 1–8) and U937 and NB4 cell lines were analyzed for lentiviral transduction with LV.B7.1 and LV.IL-2/B7.1 at an m.o.i. of 10 and
LV.IL-2 at an equivalent p24 concentration of 1 Ag/106 cells, except for Pat. 8 (+), for whom a lower p24 concentration was used. (B) The effects of increased
LV.B7.1 m.o.i. on primary AML infectivity for Pat. 6. (C) The effects of inclusion of accessory proteins (Vpr, Vpu, Vif, and Nef) in addition to Tat and Rev (using the
packaging construct pCMVDR8.2 instead of pCMVDR8.91 in LV production) on LV.B7.1 transduction in Pat. 6. B7.1 surface detection was performed with FITC-
conjugated antibody except for samples from Pats. 5, 7, and 8 (*), which were treated with PE-conjugated antibody. PE-conjugated antibody consistently excites
a higher level of fluorescence compared to FITC-conjugated antibody independent of transgene expression (data not shown). Viability index was determined by
SSC/FSC in flow cytometry by comparing the fractions of live intact cells between sample populations exposed to virus supernatant or control medium.
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FIG. 4. Allogeneic mixed lymphocyte reaction with modified primary AML
blasts. (A) Eight primary AML samples were modified with lentiviral vector
(LV.GFP, LV.B7.1, LV.IL-2/B7.1, LV.IL-2) and assessed for transgene expression
(mean values are shown for % positive cells as determined by FL-1 fluorescence
IL-2 as ng/106 cells/24 h) —, not detectable. Modified cells were then tested fo
their ability to stimulate the proliferation of allogeneic T cells compared to
unmodified cells. Unstimulated T cells were included as a control. Proliferation
of stimulated T cells is expressed relative to this control value, as Stimulation
Index (S.I.). Horizontal lines represent the mean values of eight samples. *Gene
transduction data obtained from three AML samples. (B) Proliferation o
allogeneic T cells in response to recombinant IL-2: in the absence of AML blast
(diamonds), in the presence of unmodified AML blasts (squares), and in the
presence of LV.B7.1-modified AML blasts (triangles). Proliferation is expressed
as S.I.
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sample-to-sample variation appeared to be consistentbetween the two vectors and could not be overcomeeither by applying a higher m.o.i. (up to 40) or by theadditional expression of the HIV accessory proteins(Vpr, Vpu, Vif, Nef) (Figs. 3B and 3C).
The levels of IL-2 transgene expression followingLV.IL-2/B7.1 transduction also displayed variationbetween patient samples, from 1.5 to 17.4 ng/106
cells/24 h, with samples 1, 2, 5, and 8 expressing IL-2at levels markedly higher than samples 3, 4, 6, and 7 ina pattern similar to B7.1 expression.
Infection with LV.IL-2 at equivalent m.o.i. (Pats. 1 to 7)resulted in significantly higher levels of IL-2 transgeneexpression compared with LV.IL-2/B7.1. In U937, this was118 ng/106 cells/24 h, 10-fold higher than in LV.IL-2/B7.1-infected cells, and in NB4, the increase was 25-fold, from9.5 to 238 ng/106 cells/24 h. In primary cells, the enhance-ment was between 17- and 21-fold for samples 1, 2, and 5but only 2- to 9-fold for samples 3, 6, and 7.
The infectability of AML patient samples appearedto be vector independent as all three vectors providesimilar levels of transduction efficiencies within thesame sample population. The overall levels of trans-gene expression in primary AML blasts were compara-ble to the levels observed in the established cell lines.We detected B7.1 and IL-2 transgene expression onlyfollowing gene transduction and not as a result of invitro culture or infection with the control vector,LV.GFP (Fig. 4A).
Allostimulatory Activity of Modified Primary AMLBlastsUsing a panel of eight primary AML samples, wecompared the ability of the transduced and unmodifiedAML blasts to stimulate allogeneic T cells (Fig. 4A).LV.IL-2/B7.1-modified AML blasts induced a significantincrease in allo T cell stimulation compared tounmodified or GFP-modified cells ( P = 0.002 and P =0.0056, respectively). LV.IL-2/B7.1-modified AML blastsinduced higher levels of allo T cell stimulation (mean54) than AML blasts modified with either LV.B7.1 orLV.IL-2 alone (mean 31 and 40, respectively). SinceAML blasts infected with LV.IL-2.B7.1 expressed lowerlevels of B7.1 and IL-2 than cells infected with a singlegene, this shows that the combination providedsuperior T cell stimulation than when B7.1 or IL-2was expressed alone (Fig. 4A). The enhanced T cellstimulation was partially blocked by the presence ofanti-CD80 antibody by an average of 50% for LV.B7-infected cells and an average of 40% for LV.IL-2/B7.1-infected cells (data not shown).
To investigate the possibility that the allogeneic T cellswere proliferating nonspecifically in response to IL-2, wecarried out experiments using several doses of recombi-nant IL-2 (Fig. 4B). T cells cultured with IL-2 showedproliferation in the absence of AML cells; however, this
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s
,
r
f
s
was much greater when AML cells were also present. Thisproliferation was further increased by B7.1 modification ofthe AML blasts. This suggests that although some non-specific T cell proliferation was being induced, it isunlikely that the response elicited by IL-2/B7.1 AML cellswas totally nonspecific.
Stimulation of Patients’ Peripheral BloodMononuclear Cells (PBMCs) by Modified PrimaryAML BlastsWe then assessed the ability of AML blasts to stimulatethe proliferation of the patients’ own PBMCs. We testedtwo autologous PBMC samples, one obtained from a
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ARTICLEdoi:10.1016/j.ymthe.2004.09.006
patient at diagnosis and one obtained from a patient inchemotherapy-induced remission (Patient 9). In addi-tion we tested PBMCs from two patients post-allogeneicBMT, after the establishment of donor chimerism. Fig. 5shows the levels of transgene expression and thesimulation index (S.I.) values for these patients.Although there was notable variation in the S.I. valuesin different patients, a general trend is apparent. LV.B7.1modification caused slight increases in stimulation(mean S.I. = 18) compared to unmodified cells (meanS.I. = 13). LV.IL-2 modification caused greater increasesin stimulation (mean S.I. = 140). However, the greateststimulation was achieved by AML blasts modified withLV.IL-2/B7.1 (mean S.I. = 227). As with the allogeneicMLR, this was despite lower levels of B7.1 and IL-2 than
FIG. 5. Mixed lymphocyte reaction (MLR) with modified primary AML blasts and
B7.1 infected) were tested for their ability to stimulate patients’ own PBMCs. PB
expressed as S.I. B7.1 modification is expressed as % positive cells and IL-2 as n
blasts obtained at diagnosis and PBMCs obtained after BMT with one MHC mi
obtained during chemotherapy induced remission (no BMT), Patient 5—AML b
unrelated donor, Patient 8—AML blasts and nonremission PBMCs harvested at d
blocking antibodies (anti-CD80, anti-MHC-I, and anti-MHC-II).
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in AML blasts modified to express either B7.1 or IL-2individually. Therefore, the expression of IL-2/B7.1resulted in greater stimulation of PBMCs than could beachieved by either B7.1 or IL-2 alone. Proliferation waspartially blocked by the presence of anti-CD80 antibodyby 60% for LV.B7-infected cells and 25% for LV.IL-2/B7.1-infected cells. In addition, proliferation was parti-ally blocked by the presence of anti-MHC-I but not bythe presence of anti-MHC-II (Fig. 5B).
Stimulation of Post-BMT PBMCs with LV.IL-2/B7.1AML Blasts Promotes the Expansion of Activated CD8+
Cells with Increased Secretion of Th1 CytokinesWe stimulated PBMCs from bone marrow-transplantedpatients with medium alone (unstimulated control),
patients’ PBMCs. Primary AML blasts (uninfected, LV.B7.1, LV.IL-2 and LV.IL-2/
MCs alone were included and proliferation was related to this control value,
g/106 cells/24 h. (A) MLR results from four different patients: Patient 7—AML
smatch, Patient 9—AML blasts obtained at diagnosis and autologous PBMCs
lasts obtained at diagnosis and PBMCs obtained after BMT from a matched
iagnosis. (B) MLR with PBMCs and AML cells from Patient 5 in the presence of
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unmodified AML blasts, or LV.IL-2/B7.1-modified cells.At day 0 and day 13 we determined cell numbers andanalyzed surface marker expression (Fig. 6A). PBMCscultured in medium alone or stimulated with unmodifiedAML blasts did not show an increase in numbers.Conversely, cultures stimulated with LV.IL-2/B7.1-infected AML blasts showed an approximate fourfoldexpansion in numbers. In Patient 5 a clear expansion ofCD8+ cells and an increase in CD25 and CD69 expressionwas detectable when the stimulation was with LV.IL-2/B7.1-modified cells. In Patient 7 there was an expansionof CD8+ cells in all three cultures, but activation markers
FIG. 6. Analysis of post-BMT PBMCs at day 13 after two rounds of stimulation
cocultured with medium alone (unstimulated), irradiated unmodified AML blasts,
numbers were counted and frequencies of CD4+, CD8+, CD25+, and CD69+ cel
cytokine levels in culture supernatant on day 13 were determined by cytometric
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were detected only on PBMCs stimulated with LV.IL-2/B7.1-modified AML blasts.
Analysis of Th1 and Th2 cytokines in these culturesshows that stimulation of PBMCs with LV.IL-2/B7.1 AMLblasts caused increased levels of cytokine secretion, mostnotably the Th1 cytokines: IFN-g, TNF-a, and IL-2 (Fig. 6B).
The Activated Lymphocytes Proliferate Preferentiallyin Response to Unmodified AML Blasts Compared toNormal Bone Marrow CellsWe then determined the specificity of the preactivatedPBMCs by further proliferation against two different
with patients’ own AML blasts. PBMCs from Patient 5 and Patient 7 were
or irradiated LV.IL-2/B7.1-modified AML blasts. On day 0 and day 13 viable cel
ls were determined by flow cytometry, expressed as % +ve cells (A). Th1/Th2
bead analysis, expressed as pg cytokine/5 � 105 cells (B).
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targets: the patients’ own unmodified AML blasts andbone marrow cells obtained during chemotherapy-induced remission prior to allogeneic BMT (Fig. 7).
In Patient 5 proliferation was low; however, cells didshow marginally higher proliferation in response to AMLcells compared to remission bone marrow.
In Patient 7, all three lymphocyte populationsshowed low proliferation against remission BM cells.Lymphocytes initially stimulated with unmodified cellsalso showed a correspondingly low proliferation rateagainst unmodified AML blasts. However, lymphocytespreviously stimulated with LV.IL-2/B7.1-infected cellsshowed a marked preferential proliferation againstunmodified AML blasts compared to bone marrow(S.I. = 25 compared to 1.08). This suggests that apopulation of AML-specific responder cells had beenexpanded by stimulation with the IL-2/B7.1-expressingAML blasts.
DISCUSSION
The therapeutic effects of allogeneic bone marrowtransplantation and donor leukocyte infusion, togetherwith the detection of leukemia-specific cytotoxic Tlymphocytes in myeloid malignancies [41,42], provideoptimism for the development of immune gene ther-apy strategies against AML. However, ubiquitouslyexpressed leukemia-associated or leukemia-specific anti-gens have not been identified. Therefore autologouswhole cell vaccines provide an attractive strategy forthe stimulation of immune responses in AML. Theinduction of immunological responses against AMLblasts, particularly in the context of allogeneic bonemarrow transplants, may enhance the graft versusleukemia effect or the efficacy of donor leukocyteinfusions resulting in prolonged duration of remission.
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To this end we have developed a self-inactivatinglentiviral vector for the efficient transduction ofprimary AML blasts with immune regulatory factorsB7.1 and IL-2. Important vector features contributingto this efficiency are the presence of factors thatenhance nuclear import [27,28] and RNA stability[29], as well as the use of a myeloid efficient promoterderived from spleen focus-forming virus long terminalrepeats [38]. We coupled these features with ourpreviously developed bfusageneQ strategy, which utilizedthe ubiquitously expressed endoprotease furin to proc-ess fusion proteins in the Golgi to generate thebiologically active constituents [39]. This enables themonocistronic expression of multiple proteins as asingle precursor. Therefore, we have shown for thefirst time with this vector system the genetic modifi-cation of between 40 and 99% of primary AML blastswith both B7.1 and IL-2 as a result of a single round ofinfection at an m.o.i. of 10. The expression of theencoded proteins (GFP, B7.1, or IL-2) in these cells wasat least 1 log above background. These are consistentlyhigher than the transduction efficiencies we couldpreviously achieve with multiple rounds of infectionwith retroviral [3], adenoviral, and adeno-associatedvirus vectors at substantially higher m.o.i. (unpublisheddata). The levels of lentiviral-AML transductionobserved here are superior to those previously shownfor CMV-driven constructs without the cppt/WPREenhancer combination [32,35] and comparable toCMV-driven constructs with cppt/WPRE, but at asubstantially higher m.o.i. of 50 [36]. However, withinsome primary samples there were subpopulations ofcells that appeared to be less susceptible to trans-duction (Patients 3, 4, 6, 7). This resistance wasindependent of the proteins encoded by the vector(Fig. 3A) and could not be overcome by the use of
FIG. 7. Proliferation assay showing
the response of expanded PBMCs
to the patients’ AML blasts or
remission bone marrow cells.
PBMCs from Patient 5 and Patient
7 were initially primed by coculture
with LV.IL-2/B7.1-infected AML
blasts, unmodified AML blasts, or
medium only (unstimulated), with
subsequent restimulation carried
out on days 7 and 14. On day 20
the specificity of the expanded cells
was examined by testing their abil-
ity to proliferate in response to the
patient’s unmodified AML or remis-
sion bone marrow cells in a 5 day
MLR. Unstimulated responder cells
were included as a control for each
group. Proliferation of stimulated
responders was related to this con-
trol value and expressed as S.I.
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higher m.o.i. (up to 40, Fig. 3B), the presence of HIVaccessory proteins (Fig. 3C), or the addition of deoxy-nucleosides to the virus stock or the infection mixture[43] (data not shown). The cause of this block toinfection remains unclear, but is unlikely to beassociated with virus entry because of the ubiquitouscell entry pathway through phospholipids [44] utilizedby VSV-G. In addition, the use of an amphotropicenvelope pseudotype instead of, or in addition to, VSV-G did not provide any improvement in the trans-duction rates (manuscript in preparation). Despite theability of lentiviral vectors to transduce postmitoticcells, we still found a correlation between proliferationand infectability as demonstrated by the positiveinfluence of SCF and IL-3 (up to 20% increase ininfection, data not shown). This is in agreement withfindings previously reported with noncycling T cells[45] and hematopoietic stem cells [46].
The levels of both IL-2 and B7.1 in blast cellsinfected with LV.IL-2/B7.1 were almost always lowerthan in cells infected with the single protein vectors(LV.IL-2 or LV.IL-2/B7.1). This may be the product ofreduced rates of transcription, translation, or processingof the fusion protein and the present studies do notdiscriminate between these possibilities. However, wewere able to detect IL-2 that remained bound to B7.1on the cell surface (data not shown), indicating theincomplete proteolytic cleavage of the precursor IL-2/B7.1 fusion protein. Nonetheless this is consistent withour previous findings that endogenous furin expressionvaries between tissue type [39], and despite high levelsof soluble IL-2 being secreted, a proportion remainattached to the membrane-anchored B7.1. Whether theuncleaved IL-2 attached to B7.1 acts as a novel antigenis not clear at present. However, the data presentedhere and in our previous study [39] demonstratebiological activities of B7.1 and IL-2 generated by thisstrategy. In addition, the levels of IL-2 secretion in ourlentiviral fusagene constructs remain substantiallyhigher and more stable than those achieved in IRESconfigurations (unpublished data by us and others[36]).
The efficient transduction of primary AML blastsallowed us to assess the effects of B7.1 and IL-2expression on the immunogenicity of primary AMLblasts. This was performed using lymphocytes obtainedfrom: (i) healthy donors (allogeneic), (ii) patients priorto or following chemotherapy-induced remission(autologous), and (iii) patients post-allogeneic BMTfollowing reduced intensity conditioning (chimeric).In all cases, levels of B7.1 and IL-2 were lower inAML blasts transfected with both genes than in cellstransfected with a single gene. Despite this, all theresponder cells tested showed the greatest proliferationagainst LV.IL-2/B7.1-transduced AML blasts. The levelsof autologous stimulation observed here were greater
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than those previously reported by Stripeke et al., whofirst described the use of LV transduction of primaryAML with B7.1/GM-CSF to generate whole cell vaccines[35]. Higher levels of autologous T cell stimulation,comparable to ours, have been achieved by the trans-duction of primary AML with IL-7 in combination withcytokine differentiation to a DC phenotype [47].
It is worth noting that effector cells isolated frompatients prior to induction of remission would have beenrecently exposed to a highly immunosuppressive envi-ronment due to large numbers of AML blasts [7]. Despitethis, they showed enhanced proliferation in response toLV.IL-2/B7.1-transduced cells (Patient 8).
In the autologous setting (Patients 8 and 9) the IL-2/B7.1-expressing AML blasts would have stimulated theproliferation of either nonspecific T cells or T cellsdirected against AML-associated/specific targets. Stimula-tion of nonspecific proliferation by IL-2 was investigatedin separate experiments using recombinant IL-2. Donor Tcells cultured with IL-2 showed proliferation in theabsence of AML cells, but this was much greater whenAML cells were also present. This proliferation was furtherincreased by B7.1 modification of the AML blasts (Fig.4B). Therefore as expected, IL-2 induces nonspecific T cellproliferation, but this cannot account for the entireproliferative response elicited by the IL-2/B7.1-expressingAML cells.
With lymphocytes from patients following allogeneicBMT patients (5 and 7), the expanded T cells may also bedirected against alloantigens. Our preliminary analysissuggests greater specificity of these T cells against AML-associated antigens, rather than antigenic determinantsof normal hematopoietic cells. This is indicated by thehigher proliferative response of T cells stimulated by theIL-2/B7.1-expressing AML blasts in the subsequent pres-ence of unmodified AML blasts coma compared toremission bone marrow cells (Fig. 7). Although the levelof T cell proliferation in Patient 5 was too low to showthis difference convincingly, Patient 7 show substan-tially greater stimulation in the presence of the patient’sown unmodified AML blasts than the remission bonemarrow cells (S.I. 25 vs 1). This is similar to the findingsof Mutis and colleagues, who stimulated autologous Tcells with B7.1-transfected AML cells, resulting in theexpansion of T cell clones that showed preferentialproliferation against AML cells compared to normalhematopoietic cells (immortalized B cells) [48]. We arenow examining the cytotoxic activity of the in vitrostimulated T cells against autologous unmodified AMLblasts.
The data presented demonstrate efficient transduc-tion of 40 to 99% (mean 54%, n = 12) in primary AMLblasts, resulting in high levels of IL-2 and B7.1 trans-gene expression. This is achieved by a single round ofinfection with the constructed self-inactivating lentivi-ral vector. The genetically modified AML blasts stim-
MOLECULAR THERAPY Vol. 11, No. 1, January 2005
Copyright C The American Society of Gene Therapy
ARTICLEdoi:10.1016/j.ymthe.2004.09.006
ulate proliferation of both allogeneic and autologouslymphocytes, as well as chimeric lymphocytes frombone marrow-transplanted AML patients. This stimula-tion results in expansion, primarily of CD8+ cells,upregulated expression of activation markers, andincreased secretion of Th1 cytokines. Therefore theLV.IL-2/B7.1-modified AML blasts are capable of stim-ulating the conditions required for the generation ofimmunological responses with a potential therapeuticeffect.
MATERIALS AND METHODS
Cell Culture
Culture media for 293T (DMEM), U937, K562, and NB4 (RPMI 1640) were
supplemented with l-glutamine, 10% FBS (Sigma), and penicillin G (50 U/
ml)/streptomycin (50 mg/ml).
Primary AML blasts were obtained from peripheral blood of adult
patients with high-count acute myeloid leukemia at diagnosis and prior
to initiation of chemotherapy. PBMCs were obtained from AML patients
prior to or following chemotherapy-induced remission and also from
healthy volunteers. AML blasts and PBMCs were purified by Histopaque
(Sigma) density gradient centrifugation and cryopreserved in X-VIVO 15
(BioWhittaker) with 10% DMSO and 50% human AB serum (Sigma).
Cryopreserved bone marrow samples from patients in remission were
obtained from the Stem Cell Laboratory (King’s College Hospital, London,
UK). T cells were isolated from healthy donor PBMCs with a T Cell
Negative Isolation Kit (Dynal, Oslo, Norway). All primary cells were
cultured in X-VIVO 15 medium. AML cultures were supplemented with
rhSCF (20 ng/ml) and rhIL-3 (10 ng/ml) (R&D Systems, UK). FAB
subtyping and karyotyping were carried out by the Cytogenetics Unit
(KCH, London).
Informed consent was obtained from all patients prior to collection of
primary cells.
Lentiviral Vector Construction
The improved self-inactivating lentiviral vector backbone HRVSINctwSV
was made by inserting an XhoI/XbaI fragment containing the WPRE
sequence from HRVPGKGFPWSIN-18 into HRVCMVGFPSIN (both kind
gifts from Didier Trono, University of Geneva, Switzerland), followed by
substitution of the CMV promoter with a BamHI/NotI fragment carrying
the cppt/SFFV U3 promoter from HRVSIN-cPPT-SEW (kindly provided by
Adrian Thrasher, Institute of Child Health, London, UK). The universal
backbone—HRVSINctwSV—was used to express GFP, B7.1, IL-2, and IL-2/
B7.1 (all constructs were verified by sequencing analysis, further
construction details available on request).
Lentiviral Vector Production and Titration
Lentiviral vectors were produced from transient calcium phosphate-
transfected 293T cells. Cells were cultured to 80% confluence in 150-
cm2 triple-layer flasks (Nalge Nunc) and transfected with 26 Ag
pCMVDR8.91, 14 Ag pMD.G, and 40 Ag vector construct. Virus harvests
were concentrated by an overnight centrifugation (8600g, 48C) followed
by ultracentrifugation (183,000g, 90 min, 48C). Virus pellets were
resuspended in HBSS (with 1% FBS) and stored at �808C. Titer was
determined by serial dilutions of virus inoculates on target U937 leukemia
cells and by calculating representative populations of transduced cells
analyzed 96 h after infection. This protocol produces titers of 5–7 � 108
transduction units (tu)/ml from a starting volume of 450 ml. When
correlated with HIV p24 concentration (ELISA, reagents from SAIC–
Frederick, Inc., MD, USA), approximately 107 tu/Ag p24 was observed.
This correlation was used to determine dosage loading of LV.IL-2, which
cannot be titered otherwise. Where stated, the packaging construct
pCMVDR8.2 was used instead of pCMVDR8.91 to express additional
HIV accessory proteins: Vpr, Vpu, Vif, and Nef.
MOLECULAR THERAPY Vol. 11, No. 1, January 2005
Copyright C The American Society of Gene Therapy
Lentiviral-Mediated Gene Transfer
Thawed AML blasts were cultured for 3 days in the presence of SCF and IL-
3 prior to LV infection. Target cells were plated at 5 � 105/ml (U937, K562,
NB4) or 1 � 106/ml (primary AML) and supplemented with 10 Ag/ml
DEAE dextran (Amersham Pharmacia) for viral complexing [49], which we
found to be 10-fold more efficient than Polybrene (unpublished data).
Aliquots of LV were thawed and infections performed overnight under
standard culture conditions. The following day cells were washed twice
and cultured for 72 h prior to analysis of IL-2 (ELISA) and B7.1 and GFP
(FACS) transgene expression. Except where stated, an m.o.i. of 10 was
used for primary AML infection. For comparative immunological assays,
the m.o.i. of LV.IL-2 was lowered to adjust for comparable IL-2 secretion
with LV.IL-2/B7.1.
Flow Cytometry
For analysis of GFP expression, cells were washed with HBSS, 1% FBS and
fixed in 150 Al 2% paraformaldehyde solution. For analysis of B7.1
transgene expression cells were stained with directly conjugated CD80–
FITC antibody or, where stated, CD80–PE antibody (Becton–Dickinson,
Oxford, UK). PBMCs were stained with the following conjugated anti-
bodies: CD4–FITC, CD8–FITC, CD25–FITC, and CD69–FITC (Becton–Dick-
inson). All labeling was performed with matched isotype controls for each
sample and analyzed using a FACSCalibur cytometer (Becton–Dickinson).
Analysis of IL-2 Production
IL-2 levels were determined with a DuoSet ELISA kit (R&D).
Mixed Lymphocyte Reaction (MLR)
Stimulators. Three days after LV infection, AML blasts were washed and
resuspended in fresh X-VIVO 15 medium at 5 � 105/ml, g-irradiated at 30
Gy, and seeded in flat-bottom 96-well plates at 5 � 104 per well. Where
required, remission bone marrow cells were irradiated and seeded in the
same way. To test the role of B7.1, MHC-I, and MHC-II, primary AML cells
were incubated with the anti-CD80 antibody clone l307.4; anti-HLA-DR,
DP, DQ clone TU39 (Becton–Dickinson); and anti-HLA-ABC clone W6/32
(Serotec, Oxford) at a concentration of 20 Ag/ml for 30 min prior to the
addition of responder cells. In experiments addressing nonspecific IL-2-
induced proliferation, various concentrations of recombinant human IL-2
(R&D Systems, UK) were added to the wells prior to the addition of
responder cells.
Responders. T cells from healthy volunteers (allogeneic assays) or PBMCs
from AML patients in remission (Patients 5, 7, and 9) were used. For assays
on Patient 8 responder cells were obtained from peripheral blood at the
time of diagnosis. AML blasts were removed from the sample using the
Dynal CD34 progenitor cell selection system (Dynal Biotech). In all
experiments responder cells were added at 1 � 105 per well, giving a
stimulator-to-responder ratio of 1:2. Subsets of responder cells were also
cultured alone, providing a control for unstimulated responders. MLR
cultures were incubated for 5 days. One microcurie of [methyl-3H]thymi-
dine (sp act 5.0 Ci/mmol; Amersham Pharmacia) was added to each well
for the last 16 h of culture. Incorporated thymidine was assessed by
harvesting cells onto glass fiber filters and drying them for 1 h at 808C,
and radioactivity was quantified using a Beta counter. Mean cpm values
were calculated from four wells, and the results were expressed as S.I. =
(cpm AMLs + responders)/cpm responders alone.
Analysis of Post-BMT PBMCs Stimulated with Modified AML Blasts
Post-BMT PBMCs (1 � 106) from AML patients in remission were seeded
into 3 wells of a 12-well plate, in a volume of 1 ml. LV.IL-2/B7.1-
modified or unmodified AML blasts from the same patients were
prepared at 5 � 105/ml and irradiated at 30 Gy. PBMC cultures received
1 ml of medium only (unstimulated control), unmodified AML, or LV.IL-
2/B7.1 AML. PBMC cultures were restimulated by further addition of 5 �105 irradiated AML blasts (modified or unmodified) on days 7 and 14.
Supernatants were removed on day 13 for cytokine analysis and the
lymphocytes gently resuspended and counted to assess proliferation.
129
ARTICLE doi:10.1016/j.ymthe.2004.09.006
PBMC were stained with antibodies for CD4, CD8, CD25, and CD69
expression. On day 20 cells were washed and tested for their ability to
proliferate against unmodified autologous AML or bone marrow cells in a
secondary MLR (see above).
Analysis of Cytokine Production by Stimulated PBMCs
Supernatants were removed from the cocultures described above and
stored at �208C until analysis. Levels of IFN-g, TNF-a, IL-10, IL-6, IL-4,
and IL-2 were measured with the Cytometric Bead Array Human Th1/
Th2 cytokine kit (BD Pharmingen). Supernatant from cultures contain-
ing AML blasts alone were included as a control, to subtract transgene
IL-2 released by modified AML blasts.
Statistical Analysis
Significance values were calculated using GraphPad Prism version 3.02 for
windows (GraphPad Software, San Diego, CA, USA) using a two-tailed test.
P values of less than 0.05 were taken as significant and values less than
0.005 as highly significant.
ACKNOWLEDGMENTS
We thank Mary Collins for her generosity and her assistance in our lentiviral work.
We are grateful to Dr. Adrian Thrasher (Institute of Child Health) and Dr. Didier
Trono (University of Geneva) for providing the lentiviral vectors and packaging
constructs. Our thanks also go to Michelle Kenyon for her invaluable assistance in
obtaining patient samples and to Taylor Mackey, Debbie King, and Maddy Hayes
for p24 assays. The bone marrow aspirates used in this study were provided by
KCH Stem Cell Laboratory.
RECEIVED FOR PUBLICATION MAY 20, 2004; ACCEPTED SEPTEMBER 9, 2004.
REFERENCES
1. Giles, F. J., et al. (2002). Acute myeloid leukemia. Hematology 73 – 110.
2. Galea-Lauri, J. (2002). Immunological weapons against acute myeloid leukaemia.
Immunology 107: 20 – 27.
3. Hirst, W. J., et al. (1997). Enhanced immune costimulatory activity of primary acute
myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1. Gene Ther. 4:
691 – 699.
4. Hirano, N., et al. (1996). Expression of costimulatory molecules in human leukemias.
Leukemia 10: 1168 – 1176.
5. Vollmer, M., et al. (2003). Expression of human leucocyte antigens and co-stimulatory
molecules on blasts of patients with acute myeloid leukaemia. Br. J. Haematol. 120:
1000 – 1008.
6. Whiteway, A., et al. (2003). Expression of co-stimulatory molecules on acute
myeloid leukaemia blasts may affect duration of first remission. Br. J. Haematol. 120:
442 – 451.
7. Buggins, A. G., et al. (2001). Microenvironment produced by acute myeloid leukemia
cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and
pRb pathways. J. Immunol. 167: 6021 – 6030.
8. Ando, H., et al. (2002). B7.1 immunogene therapy effectively activates CD4(+) tumor-
infiltrating lymphocytes in the central nervous system in comparison with B7.2 gene
therapy. Int. J. Oncol. 20: 807 – 812.
9. Tsuji, H., et al. (2002). Adenovirus-mediated in vivo B7-1 gene transfer induces anti-
tumor immunity against pre-established primary tumor and pulmonary metastasis of
rat osteosarcoma. Cancer Gene Ther. 9: 747 – 755.
10. Hirano, N., et al. (1997). Protective and therapeutic immunity against leukemia
induced by irradiated B7-1 (CD80)-transduced leukemic cells. Hum. Gene Ther. 8:
1375 – 1384.
11. Dunussi-Joannopoulos, K., et al. (1996). Irradiated B7-1 transduced primary acute
myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML.
Blood 87: 2938 – 2946.
12. Matulonis, U. A., et al. (1995). Role of B7-1 in mediating an immune response to
myeloid leukemia cells. Blood 85: 2507 – 2515.
13. Gaken, J. A., et al. (1997). Irradiated NC adenocarcinoma cells transduced with both
B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors. Hum.
Gene Ther. 8: 477 – 488.
14. Barnard, A. L., et al. (2000). Local versus systemic interleukin-2: tumor formation
by wild-type and B7-1-positive murine melanoma cells. Cancer Gene Ther. 7:
207 – 214.
15. Ge, N. L., et al. (2003). Prevention of hepatocellular carcinoma in mice by IL-2 and
B7-1 genes co-transfected liver cancer cell vaccines. World J. Gastroenterol. 9:
2182 – 2185.
130
16. Larchian, W. A., et al. (2000). Effectiveness of combined interleukin 2 and B7.1
vaccination strategy is dependent on the sequence and order: a liposome-mediated
gene therapy treatment for bladder cancer. Clin. Cancer Res. 6: 2913 – 2920.
17. Kochling, J., et al. (2003). Protection of mice against Philadelphia chromosome-
positive acute lymphoblastic leukemia by cell-based vaccination using nonviral,
minimalistic expression vectors and immunomodulatory oligonucleotides. Clin. Cancer
Res. 9: 3142 – 3149.
18. Hicks, C., et al. (2003). Restimulation of tumour-specific immunity in a patient
with AML following injection with B7-1 positive autologous blasts. Leuk. Res. 27:
1051 – 1061.
19. Beverly, B., et al. (1992). Reversal of in vitro T cell clonal anergy by IL-2 stimulation. Int.
Immunol. 4: 661 – 671.
20. Cortes, J. E., et al. (1999). A pilot study of interleukin-2 for adult patients with acute
myelogenous leukemia in first complete remission. Cancer 85: 1506 – 1513.
21. Roddie, P. H., et al. (2000). Gene transfer to primary acute myeloid leukaemia blasts and
myeloid leukaemia cell lines. Cytokines Cell Mol. Ther. 6: 127 – 134.
22. Wattel, E., et al. (1996). Differential efficacy of adenoviral mediated gene transfer into
cells from hematological cell lines and fresh hematological malignancies. Leukemia 10:
171 – 174.
23. Gonzalez, R., et al. (1999). Increased gene transfer in acute myeloid leukemic
cells by an adenovirus vector containing a modified fiber protein. Gene Ther. 6:
314 – 320.
24. Heinzinger, N. K., et al. (1994). The Vpr protein of human immunodeficiency virus type
1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc.
Natl. Acad. Sci. USA 91: 7311 – 7315.
25. Yao, X. J., et al. (1995). Mutagenic analysis of human immunodeficiency virus type 1
Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization
and virion incorporation. J. Virol. 69: 7032 – 7044.
26. Zufferey, R., et al. (1997). Multiply attenuated lentiviral vector achieves efficient gene
delivery in vivo. Nat. Biotechnol. 15: 871 – 875.
27. Follenzi, A., et al. (2000). Gene transfer by lentiviral vectors is limited by nuclear
translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25: 217 – 222.
28. Zennou, V., et al. (2000). HIV-1 genome nuclear import is mediated by a central DNA
flap. Cell 101: 173 – 185.
29. Zufferey, R., et al. (1999). Woodchuck hepatitis virus posttranscriptional regulatory
element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73:
2886 – 2892.
30. Barry, S. C., et al. (2001). Lentivirus vectors encoding both central polypurine tract and
posttranscriptional regulatory element provide enhanced transduction and transgene
expression. Hum. Gene Ther. 12: 1103 – 1108.
31. Yam, P. Y., et al. (2002). Design of HIV vectors for efficient gene delivery into human
hematopoietic cells. Mol. Ther. 5: 479 – 484.
32. Stripecke, R., et al. (2000). Lentiviral vectors for efficient delivery of CD80 and
granulocyte-macrophage colony-stimulating factor in human acute lymphoblastic
leukemia and acute myeloid leukemia cells to induce antileukemic immune responses.
Blood 96: 1317 – 1326.
33. Bambacioni, F., et al. (2001). Lentiviral vectors show dramatically increased efficiency
of transduction of human leukemic cell lines. Haematologica 86: 1095 – 1096.
34. Biagi, E., et al. (2001). Efficient lentiviral transduction of primary human acute
myelogenous and lymphoblastic leukemia cells. Haematologica 86: 13 – 16.
35. Koya, R. C., et al. (2002). Transduction of acute myeloid leukemia cells with third
generation self-inactivating lentiviral vectors expressing CD80 and GM-CSF: effects on
proliferation, differentiation, and stimulation of allogeneic and autologous anti-
leukemia immune responses. Leukemia 16: 1645 – 1654.
36. Stripecke, R., et al. (2003). The use of lentiviral vectors in gene therapy of leukemia:
combinatorial gene delivery of immunomodulators into leukemia cells by state-of-the-
art vectors. Blood Cells Mol. Dis. 31: 28 – 37.
37. Zufferey, R., et al. (1998). Self-inactivating lentivirus vector for safe and efficient in vivo
gene delivery. J. Virol. 72: 9873 – 9880.
38. Baum, C., et al. (1995). Novel retroviral vectors for efficient expression of the
multidrug resistance (mdr-1) gene in early hematopoietic cells. J. Virol. 69:
7541 – 7547.
39. Gaken, J., et al. (2000). Fusagene vectors: a novel strategy for the expression of multiple
genes from a single cistron. Gene Ther. 7: 1979 – 1985.
40. Liu, M. L., et al. (1996). Pseudotransduction of hepatocytes by using concentrated
pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)–Moloney murine
leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic
vectors for hepatic gene transfer. J. Virol. 70: 2497 – 2502.
41. Molldrem, J., et al. (1996). Targeted T-cell therapy for human leukemia: cytotoxic T
lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human
myeloid leukemia cells. Blood 88: 2450 – 2457.
42. Gao, L., et al. (2000). Selective elimination of leukemic CD34(+) progenitor cells by
cytotoxic T lymphocytes specific for WT1. Blood 95: 2198 – 2203.
43. Ravot, E., et al. (2002). High efficiency lentiviral gene delivery in non-dividing cells by
deoxynucleoside treatment. J. Gene Med. 4: 161 – 169.
44. Carneiro, F. A., et al. (2002). Membrane recognition by vesicular stomatitis virus involves
enthalpy-driven protein–lipid interactions. J. Virol. 76: 3756 – 3764.
MOLECULAR THERAPY Vol. 11, No. 1, January 2005
Copyright C The American Society of Gene Therapy
ARTICLEdoi:10.1016/j.ymthe.2004.09.006
45. Verhoeyen, E., et al. (2003). IL-7 surface-engineered lentiviral vectors promote
survival and efficient gene transfer in resting primary T lymphocytes. Blood 101:
2167 – 2174.
46. Sutton, R. E., et al. (1999). Transduction of human progenitor hematopoietic stem cells
by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J. Virol.
73: 3649 – 3660.
47. Bello-Fernandez, C., et al. (2003). Retrovirus-mediated IL-7 expression in leukemic
MOLECULAR THERAPY Vol. 11, No. 1, January 2005
Copyright C The American Society of Gene Therapy
dendritic cells generated from primary acute myelogenous leukemias enhances their
functional properties. Blood 101: 2184 – 2190.
48. Mutis, T., et al. (1998). CD80-transfected acute myeloid leukemia cells induce primary
allogeneic T-cell responses directed at patient specific minor histocompatibility
antigens and leukemia-associated antigens. Blood 92: 1677 – 1684.
49. Themis, M., et al. (1998). Enhanced in vitro and in vivo gene delivery using cationic
agent complexed retrovirus vectors. Gene Ther. 5: 1180 – 1186.
131