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Cytokine Therapy With Gene-Transfected Cells: Single Injection of


Irradiated Granulocyte-Macrophage Colony-Stimulating
Factor-Transduced Cells Accelerates Hematopoietic
Recovery After Cytotoxic Chemotherapy in Mice
By Felicia M. Rosenthal, Reinhard Fruh, Reinhard Henschler, Hendrik Veelken, Peter Kulmburg, Andreas Mackensen,
Bernd Gansbacher, Roland Mertelsmann, and Albrecht Lindemann

Development of cell-based delivery systems that can release CSFICMS5# 6 cells)into mice, GM-CSF serum levels of 405f
therapeutic levelsof hematopoietic growth factors into the 58 pgImLand 1832 36 pgImL were measured, respectively.
systemic circulation would facilitate treatment of patients Serum levels were comparable with levels detected 3 hours
requiring cytokine therapy. In this study, we have investi- afterinjection of100 ng recombinantmurineGM-CSF
gated the potential of granulocyte-macrophage colony-stim- (rmGM-CSF) subcutaneously (90 pg/mL). Injectionof N21
ulating factor (GM-CSF)-secreting,irradiatedsyngeneic mu- CMVGM-CSFICMS5 # 6 cells in cyclophosphamide-treated
rine cells to accelerate hematopoietic recovery after mice was as effective in accelerating neutrophil recovery as
cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma twice dailysubcutaneousinjectionsofrmGM-CSF.These
cells, were transduced with a retroviral vector containing data suggest that irradiated hematopoietic growth factor-
the murine GM-CSF cDNA. Transduced cells secreted 38 ng secreting cellsmight offer an alternative to parenteral injec-
GM-CSFI10 cells in 24 hours. After irradiation,in vitro GM- tions of recombinant cytokines in the treatment of neutro-
CSF production initially increasedup to fivefold and was penic patients.
measurable for about 2 weeks. One and 2 days after injection 0 1994 by The American Society of Hematology.
of irradiated, GM-CSF-secreting CMS-5 cells (NPICMVGM-

H EMATOPOIETIC GROWTH factors have been shown


to accelerate bone marrow (BM) recovery after che-
motherapy and radiation therapy in mice.7 Inhumans, gran-
genes into tumor cells to induce antitumor immune responses
or into hematopoietic cells to study the controls modulating
self-renewal and differentiation of hematopoietic ~ e l l s . ~ ~ - ~
ulocyte colony-stimulating factor (G-CSF) and granulocyte- Neutropenia after cytotoxic therapy is only transient, and
macrophage colony-stimulating factor (GM-CSF) are widely thus, requires cytokine substitution for a limited time period
used to shorten the phase of neutropenia after cytotoxic ther- only. Tani et alZ8have designed a subcutaneous diffusion
apy and after BM transplantation.82However, serum half- chamber apparatus for cytokine-producing fibroblasts that
life of these cytokines is relatively short (eg, 35 minutes for allows regulation of cytokine secretion either by reimplanta-
GM-CSF after interperitoneal injection in mice) and continu- tion of additional cells into the chamber or by removing
ous parenteral administration, eg, by the use of Alza minios- cells through ethanol treatment of the chamber. Ideally, gene
motic pumps, or repetitive injections, has been used to main- expression would be regulated at the transcriptional level,
tain biologically effective serum concentrations.I5Methods eg, by the use of inducible promoters. Here we propose the
to provide sustained cytokine serum levels for several days use of irradiated cytokine-gene-transduced cells that have
without the need for daily injections or surgical implantation lost the capability to proliferate in vivo, but have retained
of delivery devices would be desirable. Autologous or allo- the ability to secrete biologically active levels of cytokines
geneic cells, genetically modified to constitutively secrete for several days to weeks. Moreover, irradiation would be
the cytokine of interest, could fulfill these criteria. Several a precaution when cells are injected in vivo, as malignant
investigators have already described the successful introduc- transformation of genetically engineered fibroblasts has been
tion of genes, coding for enzymes like al-antitrypsin or described after in vivo implantation in mice.
hormones like insulin, into murine fibroblasts or myoblasts, In this report, we show acceleration of hematopoietic re-
and the therapeutic application of these cells in animal defi- covery after injection of irradiated GM-CSF-transduced
ciency models.z4 Other groups have introduced cytokine murine fibrosarcoma cells in cyclophosphamide-treated
mice.
Fromthe Departments of Medicine I (Huematology/Oncology)
MATERIAL AND METHODS
and Surgery, University Medical Center Freiburg, Feiburg, Ger-
many; and the Department of Medicine, Memorial Sloan-Kettering Retroviral vector design and conversion of vectors into viruses.
Cancer Center, New York, NY. Cloning of retroviral vectors NZICMVGM-CSF and DCIADIRIGM-
Submitted March 16, 1994; accepted July 5, 1994. CSF has been described p r e v i ~ u s l y Retroviral
.~~ vector constructs
Supported by a grant from the Deutsche Forschungsgemeinschafi were converted to the corresponding virus by electroporating vector
(Sonderforschungsbereich 364). DNA into the helper-free, ecotropic packaging cell line (GP+E-
Address reprint requests to Feliciu Rosenthal, MD, University 86)? Colonies were isolated by G418 selection (0.7 m@& of
Medical Center Freiburg, Department of Internal Medicine l, Hae- Geneticin; GIBCO Laboratories, Grand Island, NY), expanded to
matology/Oncology, Hugstetter Strasse 55, 79106 Freiburg, Ger- producer cell lines and cell free supernatant was tested for viral titer.
many. Cell line and infection of tumor cells. CMS-5 is a methylcholan-
The publication costs of this article were defrayed in part by page trene-induced, nonmetastatic fibrosarcoma cell line of BALBIc ori-
charge payment. This article must therefore be hereby marked gin. Cells were cultured in Dulbeccos modified Eagles medium
advertisement in accordance with 18 U.S.C. section 1734 solely to supplemented with 10% fetal calf serum, 100 U/mL of penicillin,
indicate this fact. 100 pg/mL of streptomycin and 2 mmoVL L-glutamine at 37C in
0 1994 by The American Society of Hematology. 5% CO2 atmosphere. Virus-producer cell lines secreting high titer
0006-4971/94/8409-0023$3.00/0 of virus (1 to 5 X IO5) were usedto infect CMS-5 cells. Bulk-

2960 Blood, Vol 84, No 9 (November l ) , 1994: pp 2960-2965


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THERAPY WITH GENE-TRANSFECTEDCELLS 296 1

infected cells were isolated by G41 8 selection, expanded to cell lines Table 1. GM-CSF Secretion by Transduced CMSS Bulk Culturesaa
and used for further analysis. Daerminod by ELBA
Cyrokine assay. Secretion of GM-CSF into supernatants by ret- Virus Clone GM-CSF Ing/lO' cells and 24 h)
rovirally infected tumor cells and GM-CSF serum levels were deter- ~ _ _ _ _ _ ~

mined using an appropriate bioassay and confirmed by enzyme- N2lCMVGM-CSFE86 # 6 128 t- 6.4
linked immunosorbent assay (ELISA; Endogen, Boston, MA). N21CMVGM-CSFE86 # 10 80 2 1.4
Supernatant from semiconfluent irradiated or unirradiated parental NZlCMVGM-CSFE86 # l ? 126 t- 1.8
or cytokine-secreting CMS-5 cells was collected after 24 hours, and DC/ADlRlGM-CSFE86 # 4 16 t- 2.5
assayed for GM-CSF production after determining the number of DC/AD/RlGM-CSFiE86 # 15 8 2 3.4
viable cells. Irradiation was performed at room temperature with a DCIADIRIGM-CSFlE86 # 17 25 t- 0.1
'37Cs-irradiationsource at a dose-rate of 3 Gy/min. Irradiated (35, DCIADIRIGM-CSFIE86# 18 21 2 3.6
50, and 100 Cy) CMS-5 andN2/CMVGM-CSF/CMS5 # 6 cells Data are represented as means of duplicate determinations t- SE.
(lo6) were plated in 25-mL tissue-culture flasks and the amount of
GM-CSF in a 24-hour culture supernatant l , 3,6,9, or 12 days after
irradiation was determined. GM-CSF biologic activity was measured
by testing the ability of GM-CSF-containing preparations to induce
GM-CSF secretion after irradiation of cytokine-gene-
'H-thymidine incorporation into DNA of GM-CSF-dependent mu- transducedjibrosarcoma cells. To prevent proliferation of
rine 3 2 x 1 3 cells as previously de~cribed.2~ cytokine producing cells in vivo, cells were irradiated before
Treament of mice. Female BALB/c mice were purchased from injection. To study how long cytokine secretion would con-
the Zentralinstitut f i r Versuchstierzucht in Hannover, Germany and tinue after irradiation, N2/CMVGM-CSF/CMS5 # 6 cells
used at 9 to 11 weeks old. On day 0 and day 2, all mice received were irradiated with 35, 50, and 100 Gy, respectively, and
150 mg/kg of cyclophosphamide interperitoneally. One group of plated at a density of lo6cells/25-mL tissue-culture medium.
mice did not receive further treatment, another group was injected GM-CSF levels in 24-hour culture supernatants were deter-
subcutaneously with 100 ngof recombinant murine GM-CSF mined 1, 3, 6, 9, and 12 days after irradiation. Unirradiated
(rmGM-CSF; Boehringer Mannheim, Germany) twice daily on days
N2/CMVGM-CSF/CMS5 # 6 cells produced 38 ng/106 cells
3 through 10. This dose of rmGM-CSF has previously been shown
to accelerate hematopoietic recovery after chemotherapy in mice.'
in 24 hours (Fig 1, day -1 before irradiation). After irradia-
The last group of mice was injected subcutaneously on day 3 with tion, cytokine secretion initially increased threefold to five-
a total of lo7irradiated N2lCMVGM-CSFlCMS5 # 6 cells, split into fold depending on the dose of irradiation applied. GM-CSF
two injection sites. From day 4, blood was drawn daily from lateral levels decreased to levels similar to those observed before
tail veins into EDTA-coated microvette tubes (Sarstedt, Heidelberg, irradiation by day12.As the number of viable cells de-
Germany). After lysis of erythrocytes with Turk solution, absolute creased continuously, these data suggest that biologically
leukocyte counts were determined in a Neubauer Chamber. Differen- active GM-CSF is also released by radiation-damaged cells
tial counts were performed every other day. GM-CSF serum levels for several days.
were determined on days 1 through 4 after injection of GM-CSF- GM-CSF serum levels after injection of GM-CSF-secre-
producing cells.
tingjibrosarcoma cells in BALB/c mice. To obtain informa-
Statistical analyses. Mean ? SE was calculated. Differences
between groups were examined for statistical significance by a two- tion about the time course of GM-CSF serum concentrations,
tailed, two-sample t-test. A P value s .05 was considered to indicate blood was drawn at different time points after subcutaneous
statistical significance. injection of irradiated N2/CMVGM-CSF/CMS5 # 6 cells or
3 and 10 hours after subcutaneous injection of rmGM-CSF.
GM-CSF serum levels were determined in a bioassay and
RESULTS
confirmed by ELISA. In Fig 2A, GM-CSF serum levels of
Infection ofjibrosarcoma cells. The structures of the ret- mice injected subcutaneously with 100 ng rmGM-CSF are
roviral vectors used to transduce the murine fibrosarcoma shown 3 and 10 hours after injection. Three hours after injec-
cell line CMS-5 have been described previou~ly?~ Virus- tion of rmGM-CSF, a serum level of 93 t 1 1 pg/mL was
containing, cell-free supernatant of infected packaging cell measured, whereas at 10 hours, serum GM-CSF levels were
clones was used to transduce CMS-5 cells. G418-resistant below the limit of detection. Figure 2B shows GM-CSF
CMS-5 bulk-infected cells were screened for GM-CSF ex- serum levels 1 to 4 days after injection of lo7irradiated N2/
pression by analyzing GM-CSF release into the culture su- CMVGM-CSFfCMS5 # 6 cells, which is 3 to 6 days after
pernatant. Parental CMS-5 cells did not secrete any detect- initiation of cytotoxic therapy. One and 2 days after injection
able GM-CSF in either assay. G418-resistant cytokine gene of GM-CSF-secreting cells, GM-CSF serum levels of 405
transduced cells differed in their ability to synthesize and t 58 pg/mL and 183 2 36 pg/mL were measured, respec-
secrete GM-CSF, depending on the retroviral vector con- tively. On days 3 and 4, GM-CSF serum levels were 88 2
struct and the virus-producer clone used to transduce target 24 pg/mL and 82 t 26 pg/mL, respectively, comparable
cells (Table 1). GM-CSF secretion of bulk-infected fibro- with the level detected 3 hours after subcutaneous injection
sarcoma cells transduced with the GM-CSF vector ranged of 100 ng rmGM-CSF. NoGM-CSF was found in the serum
from 0 to 128 ng/106 cells in 24 hours. GM-CSF production of mice injected with the same number of parental, nontrans-
of cells was found to decrease after extended periods of duced CMS-5 cells.
culture without G418 selection. Bulk-infected NUCMVGM- Hematologic changes in cyclophosphamide-treated mice,
CSF/CMSS # 6 cells that were used for the in vivo studies after injection of GM-CSF-secreting fibrosarcoma cells or
described here, secreted 38 ng/106 cells in 24 hours unless twice daily injections of recombinant GM-CSF. Nine-to
irradiated (see below). 1l-week-old female BALB/c mice were injected intraperito-
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ROSENTHAL ET AL

1 T

t 1

-1 1 3 6 B 1 2 -1 1 3 6 B 1 2 -1 1 3 6 0 1 2

Dlya Pl0.r ImdMkn


Fig 1. GM-CSF production by W/CMVGM-CSF/CMSL # 6 CMS-5 cells and cell survival efter irradiation. One million cells were irradiated
on day 0 with 35 (A). 50 (B). or 100Gy (C), respectively, and platedin 25-mL tissue-cutture flasks. GM-CSF levels in 24-hour culture supernantants
were determined by bioassay on days -1 (before irradiation) anddays 1,3,6,9, and 12 after irradiation and after supernatant collection cells
were trypsinized andcounted. Values are means k SE of six (GM-CSF) or two (cell number) determinations.

neally on days 0 and 2 with l50 mgkg cyclophosphamide, BALB/c mice were between 9,000 and lO,OOO/pL. Three
the control group didnot receive further treatment. One days after initiation of cyclophosphamide therapy leukocyte
group of mice was injected subcutaneously with IO' irradi- counts dropped to nadir levels of 1,000 to 1,5OO/yLin all
ated N2/CMVGM-CSF/CMSS # 6 cells on day 3, another mice, persisting until day 6. Starting from day 7, a difference
group was injected twice daily (d3-d10) subcutaneously with in absolute leukocyte counts between GM-CSF-treated mice
100 ng of rmCM-CSF. Figure 3 shows hematologic changes and control mice injected with cyclophosphamide only was
in BALB/c mice after therapy. Baseline leukocyte counts in noted. On day 8, absolute white blood cell (WBC) counts

I n IWd. CM86 RC.


400- 4a!

a- sm

m- a00
Fig 2. GM-CSF serum levels in BALBlc miceafter
injection ofGM-CSF-secreting CMS-5 cells, parental
CMS-5 cells or recombinant murine GM-CSF. (A) GM-
CSF serum levels 3 and 10 hours aftersubcutaneous
rm 1m
injection of 100 n g rmGM-CSF. (B} GM-CSF serum
levels 1.2.3, and 4 days after injection of IO' irradi-
ated (35 Gyl WICMVGM-CSFICMS5 # 6 cells or un-
transduced CMS-5 cells. Values are means 2 SE of
0 0 fourmicefrom two different experiments (days 1
a 10 i 2 a 4
and 2, and 10 hours) or 2 mice (days 3 and 4, and 3
Dsys pon InjsUlon hours). nd, not done; >, below limit of detection.
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THERAPY WITH GENE-TRANSFECTEDCELLS 2963

Fig 3. Peripheral WBC countsincyclophospha-


mide-treated BALBlcmice, after subcutaneous injec-
tion of rmGM-CSF or irradiated GM-CSF-secreting
*
fibrosarcoma cells. Rwulta represent means SE
of five mice from two dlfferent experiments (days3
through 81 and two mice (days9 and 10). Subcutane-
ous injmtiona of rmGM-CSF or NZ/CMVGM-CSF/
CMS5 I 6 cella are illustrated by arrows. ( P values
for day 8 cydophosphemide v GM-CSF subcutane-
ously: P = .010; cyclophosphemide v NZICMVGM-
CSFlCMS5 m P = ,036).

in mice injected subcutaneously twice daily with rmGM- smear examinations showed absolute neutropenia (<100/
CSF as well as in mice treated with a single injection of pL) in all treatment groups (data not shown). Onday 7,
irradiated GM-CSF-secreting syngeneic cells were signifi- mice treated with subcutaneous rmGM-CSF or GM-CSF-
cantly higher as compared with control mice (cyclophospha- secreting cells showed almost twice the absolute leukocyte
mide v GM-CSF subcutaneously: P = .010,cyclophospha- levels of control mice, accounted for by a higher proportion
mide v NUCMVGM-CSFICMSS#6: P = .036). Average of myeloid cells. Baseline myeloid counts were reached in
WBC counts on day 7 were 4,38O/pL in the cytokine treat- both GM-CSF-treated populations 7 days after the first cy-
ment group compared with 3,005/pL in control mice. On clophosphamide injection or 4 days after initiation of cyto-
day 8,WBC count of the cytokine treatment group had in- kine treatment, respectively, whereas control mice had only
creased to 9,26O/pL, and in controls to 6,34O/pL. In mice reached approximately half-normal values at that time. No
treated with rmGM-CSF subcutaneously or with GM-CSF- significantdifferencesin absolute lymphocyte counts were
secreting cells, leukocyte counts continued to increase until noted between different treatment groups. On8,day the differ-
day 9 and returned toward baseline levels on day 10. After ence in absolute myeloid cells between cyclophosphamide-
recovery of hematopoiesis (eg, after day 9) no siginficant only-treated mice and those with addition of hematopoietic
difference in absolute leukocyte counts is e ~ p e c t e d . ~ growth factor treatment was even more pronounced (cyclo-
Table 2 shows differential W C counts of treatment phosphamide v GM-CSF subcutaneously: P = .019, cyclo-
groups as absolute values of leukocyte subpopulations on phosphamide v NUCMVGM-CSFICMS5 M: P = .W).
days 7 and 8 after initiation of treatment. Untreated BALB/
DISCUSSION
c mice had a differential count of about 400 monocytes,
1,800 granulocytes and 7,100 lymphocytes. On day 3 The use of recombinant hematopoietic growth factors has
through 5 , ie, 1 to 3 days after cytotoxic therapy, blood reduced morbidity of cytotoxic chemotherapy in tumor pa-
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2964 ROSENTHAL ET AL

Table 2. Leukocyte Subpopulations

Control Cyclophosphamide + GM-CSF + GM/CMS5 U6


Day 7
Lymphocytes 7,100 t 350 1,674 t 102 1,869 i 261 1,465 t 304
Myeloid cells 2,200 i 480 1,331 t 415 2.771 i 438 2,655 t_ 508
Day 8
Lymphocytes 7,100 t 350 3,410 t 316 4,078 i 651 2.977 i 638
Myeloid cells 2,200 +- 480 3,657 t 163 5,304 2 310 6,713 f 802
Results are shown for untreated control animals, and for days 7 and 8 after cyclophosphamide administration and subcutaneous injection of
rmGM-CSF or irradiated GM-CSF-secreting syngeneic NP/CMVGM-CSF/CMSB #6 cells (GM/CMS #6).Values are means SE of five mice from
two different experiments. (Pvalues for day 8, myeloid cells: cyclophosphamide v subcutaneous GM-CSF: P = .019, cyclophosphamide v N2/
CMVGM-CSF/CMS5#6 P = ,044).

tients by amelioration of neutropenia and its associated infec- Tani et alZ8or miniosmotic pumps are likely to be equally
tious complications.82Currently, growth factors are applied effective, both approaches require surgical interventions.l4,l5
by daily subcutaneous injections or continuous intravenous With current technology, it is unlikelythat cytokine-gene-
infusion. In this study, we show that a single injection of transfected cells would replace administration of recombi-
irradiated GM-CSF-transduced murine fibrosarcoma cells nant growth factors in transient, chemotherapy-induced neu-
is as effective in accelerating hematopoietic recovery after tropenias except in special circumstances when poor patient
cyclophosphamide chemotherapy as twice daily subcutane- compliance or other factors make daily administration diffi-
ous injections of recombinant GM-CSF. cult or impossible. However, successful treatment of chronic
Because long-term expression of GM-CSF is not desirable neutropenic disorders like Kostmanns syndrome (congenital
for the treatment of chemotherapy-induced neutropenia, non- neutropenia) and cyclic or idiopathic neutropenias with ge-
proliferating cells with a finite life span should be used. netically engineered cells or slow-release formulations of G-
Irradiation of transfected cells is one option and has the CSF might be possible in the nearfut~re.~. In this situation,
additional advantage of preventing outgrowth of possibly the application of G-CSF gene-transfected unirradiated, non-
transformed cells in vivo.28Here we show that after irradia- proliferating autologous cells, possibly in conjunction with
tion of GM-CSF gene-transduced cells with up to 100 Gy, a suicide gene allowing interruption of cytokine secretion in
cytokine production persisted for more than 12 days in vitro case of detrimental effects or other clinical indications, ap-
(Fig 1). After irradiation, GM-CSF secretion initially in- pears to bea promising strategy, and is currently under inves-
creased. This effect could possibly be caused by cytokine tigation in our laboratory.zz~23~36
leakage from radiation-damaged cells or by radiation-in-
duced activation of transcription factors.*, Irradiated cells ACKNOWLEDGMENT
were incapable of further proliferation in vitro, as shown by We thank Carolin Heifer for excellent technical assistance and
decreasing cell numbers over time. There was no evidence Claudia Schmoor for help with statistical analyses.
of tumor growth or other side effects in mice observed for
up to 2 months after treatment (data not shown). REFERENCES
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From www.bloodjournal.org by guest on July 14, 2017. For personal use only.

1994 84: 2960-2965

Cytokine therapy with gene-transfected cells: single injection of


irradiated granulocyte-macrophage colony-stimulating
factor-transduced cells accelerates hematopoietic recovery after
cytotoxic chemotherapy in mice
FM Rosenthal, R Fruh, R Henschler, H Veelken, P Kulmburg, A Mackensen, B Gansbacher, R
Mertelsmann and A Lindemann

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