0022-5347199/16134960$03.

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THE JOLWAL OF UROLOGY Vol. 161, 960-963, March 1999
Copyright 8 1999 by AMERICAX UROLOCICAL
ASSOCIATION,1h.C Printed in U.S.A.

NEUTRALIZING ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR

ANTIBODY INHIBITS FURTHER GROWTH OF ESTABLISHED
PROSTATE CANCER AND METASTASES IN A PRE-CLINICAL MODEL
OSTAP MELNYK,* MICHAEL ZIMMERMAN, K. JIN KIM AND MARC SHUMAN
From the Cancer Research Institute, University of California at San Francisco and Genentech, Inc., South Son Francisco, San Francisco,
Ca 1ifornia

ABSTRACT
Purpose: The formation of new blood vessels from the pre-existing vasculature is necessary for
support of primary tumor growth and appears coincident with the development of metastasis. In
previous studies, inhibition of vascular endothelial growth factor (VEGF), a potent angiogenic
factor and mediator of vascular permeability, inhibited tumor neovascularization with conse-
quent inhibition of both primary tumor growth and micrometastases when administered a t the
time of tumor inoculation. In the present study, we examined the effect of inhibiting VEGF on
primary tumor growth and metastases in a n in vivo model of established metastatic prostate
cancer.
Materials and Methods: The human prostate cancer cell line DU-145 was found to secrete
VEGF. DU-145.luciferase, a subclone stably transfected with a n expression vector encoding the
luciferase gene, injected subcutaneously, consistently formed tumors in C.B.-17 scid lscid mice.
After 6 weeks, assay of whole lung lysates showed significant luciferase activity, consistent with
the presence of micrometastasis.
Results: Twice weekly treatment of the animals with a monoclonal anti-VEGF neutralizing
antibody, A4.6.1, not only suppressed primary tumor growth, but inhibited metastatic dissemi-
nation to the lung. When treatment was delayed until the primary tumors were well-established,
further growth was still inhibited, as was the progression of metastatic disease.
Conclusion: Inhibition of tumor-secreted VEGF by a neutralizing antibody is sufficient to
significantly impair prostate tumor growth and its subsequent metastasis in a n in vivo model of
established advanced prostate cancer, These data suggest a critical role for VEGF in initiation
and maintenance of tumor angiogenesis in prostate cancer. Inhibition of VEGF in patients with
VEGF-secreting prostate cancers may prove a n effective approach for inhibiting disease progres-
sion even after micro-metastatic dissemination has occurred.
KEY WORDS:VEGF, angiogenesis, metastasis, prostate cancer, DU-145
VEGF is a unique endothelial cell-specific mitogen and MATERIALS AND METHODS
potent inducer of vascular permeability.'.* VEGF is ex- Antibodies. The following murine monoclonal antibodies
pressed a t low levels in the quiescent vasculature of normal were used: A4.6.1, a neutralizing anti-VEGF antibody raised
tissues such as kidney and brain,3 suggesting a physiologic against recombinant human VEGF" with demonstrated in-
role in normal endothelial cell turnover and vascular main- hibitory activity in tumor xenograft models and, as controls,
tenance. In addition, VEGF appears to be a critical factor for either 6E10, an antibody of the same isotype (IgG1) directed
the promotion of tumor angiogenesis: VEGF is expressed a t against the irrelevant epitope gp120, a viral coat protein, or
high levels in a broad spectrum of malignancies, including
vehicle only (PBS). In other published data,g 6E10 and PBS
carcinoma of the breast: ovary: and brain.7 In ex-
proved equivalent with neither showing inhibitory activity
perimental systems, inhibition of VEGF decreases tumor
neo-vascularization and substantially inhibits primary tu- against either primary tumor growth or metastases.
mor growth and Vector construct. The luciferase expression vector
In prostate cancer, the degree of tumor vascularization pRK7.luciferase was constructed by excising the luciferase
correlates with the development of metastatic disease." gene with Hind I11 and Sma I from pT3/T7-luc (Clontech,
Though the critical factors that promote angiogenesis in Inc.) and ligation into pRK7 (Genentech, Inc.), an expression
prostate cancer are yet to be defined, we hypothesized that vector which contains both the human cytomegalovirus pro-
VEGF may promote tumor angiogenesis in prostate cancer. moter and enhancer as well as the SV40 polyadenylation
Utilizing a VEGF-neutralizing monoclonal antibody," we signal. This vector was co-transfected with pRK5.neo (Ge-
examined the effect of VEGF inhibition in an in vivo model of nentech, Inc.), a similar vector conferring neomycin resis-
metastatic prostate cancer. We found that inhibition of tance.
VEGF suppresses both primary prostatic tumor growth and Cell line. DU-145, a hormone independent and weakly acid
dissemination of micrometastasis. In addition, it appears to phosphatase-positive human prostate carcinoma cell line,"
inhibit further growth and metastatic progression of well- was obtained from the American Type Culture Collection and
established tumor grafts. maintained in DME H-16m-12 a t 1:1v/v supplemented with
Accepted for publication September 24,1998. 10% fetal bovine serum. Transfection was performed by the
* Requests for reprints: Cancer Research Institute, UCSF School calcium phosphate method. Transfectants were selected with
of Medicine, Box 0128,San Francisco CA 94143-0128. the neomycin analog G418 (400 pg./ml., GIBCOBRL). DU-
960
 INHIBITION OF VEGF SUPPRESSES PROSTATE CANCER PROGRESSION 96 1
145.luciferase was obtained by co-transfection of DU-145 ictivity; thus, lung luciferase activity identifies eatablished
with pRK7.luciferase and pRK5.ne0, selection in G418 to nicro-metastatic foci within the lung rather than the incidental
obtain stable transfectants, and then screening for luciferase ransit of luciferase-positive tumor cells through the organ in
expression. *dating bl00d.l~
VEGF ELZSA. 1 X lo5 DU-145.luciferase cells were grown To study the effect of VEGF inhibition on primary tumor
in 1.0 ml. serum-free culture media on 24-well tissue culture !powth in vivo, 2 x lo6 DU-145.luciferase cells were injected
plates. Conditioned media was collected after 7 days, and the into the flank of SCID mice. Anti-VEGF therapy was initi-
concentration of secreted VEGF was measured by ELISA, as ated on day 3. With early initiation of treatment, significant
described. l3 inhibition of primary tumor growth was noted in the anti-
Primary tumorigenesis and metastasis model. Six weeks VEGF antibody-treated animals compared with contmls (fig.
old female C.B.-17 scidlscid mice were maintained in a 1).This difference in tumor size was evident at day 22 and
pathogen-free environment throughout the experiment. Mice persisted for the duration of the experiment. On day 42 the
were injected subcutaneously in the flank with 2 X lo6 DU- average tumor size was 57 ? 28 mm.3 in mice treated with
145.luciferase tumor cells (dissociated from stock plates with 100 pg. of A6.4.1 i.p. bi-weekly, 212 2 111 ~ n m in . ~mice
trypsin, washed and resuspended in 0.1 ml. PBS). Antibody treated with 10 pg. bi-weekly, versus 449 2 97 mm.3 in
A4.6.1 was injected intraperitoneally (i.p.) at a dose of either controls (p = 0.0017 and 0.13 respectively).
10 Fg. or 100 pg. twice weekly in a volume of 0.2 ml. In initial On day 42 the animals were sacrificed and the lungs ana-
experiments, treatment was initiated within one week after lyzed for luciferase activity The average metastatic activity
cell injection. Control mice were treated with 100 pg. of mAb in the control group measured 1.53 ? 0.97 RLFU per mg. of
6E10. Each treatment group was comprised of 8 mice. Tumor whole organ lysate (background levels = 0.025 RLFU), while
sizes were determined weekly by multiplying their width by neither group treated with A6.4.1 showed luciferase activity
their length. At 6 weeks after tumor cell inoculation, animals above background (0.0092 0.13 and 0.011 2 0.02, respec-
were sacrificed and primary tumors were excised and tively), (fig. 2). Thus, while 5/8 control mice demonstrated
weighed. Necropsies were performed and organs were har- micrometastasis, 0/16 anti-VEGF treated animals showed
vested for luciferase assay. evidence of metastasis as measured by whole organ lucif-
Metastasis assay. Metastasis of luciferase-expressing tu- erase activity (Fisher’s exact p-value = 0.0013).
mor cells was determined by luciferase assay of whole organ Subsequent experiments were designed to determine
lysates. Whole organs were first pulverized following fixation whether delaying treatment with anti-VEGF therapy re-
in liquid nitrogen. Luciferase assays were performed as per mains effective if initiated a h r the primary xenograft has
manufacturer’s instructions using a commercial luciferase become well-established. Therefore, therapy with 100 pg.
assay kit (Promega). Samples were incubated and rocked in bi-weekly of mAb A4.6.1 was delayed until 4 weeks after
Cell Culture Lysis Reagent (Promega), 2: 1 w/v samp1e:lysis
buffer, for 90 minutes at 4C. 20 pl. of lysed sample was then
added to 100 pl. of re-hydrated Assay Substrate (Promega),
with photon emission measured by luminometry. Metastasis, 700
measured as luciferase activity in organ lysates and ex-
pressed in Relative Light-Forming Units (RLFU), was quan- U low dose-10 mcg.
tified against standards of whole organ lysates to which --[I high dose-1 00 rncg.
600
known numbers of luciferase-expressing tumor cells had
been added.
Statistical analysis. All statististical analysis was per-
formed using Abacus Concepts, StatView software (Abacus 500
Concepts, Inc., Berkeley, CA, 1994). Data are expressed as
means ? standard errors. Differences in tumor size between hl
treated and control groups were evaluated by unpaired t E
tests. Metastases, scored as present or absent, were evalu- E 400
ated by two-way contingency tables with Fisher’s Exact Test Lu
applied. Differences in metastatic activity, quantitated as N,
cn
luciferase activity in RLFU, were assessed by the nonpara- U
metric Mann-Whitney U test given the presence of extreme 0 300
outliers in the untreated group. fI-
RESULTS 20c
The human prostate cancer cell line DU-145 was found to
secrete VEGF in a concentration of 11 ng./ml. by ELISA,
comparable to levels in other types of tumor cell lines.’.’ The
1O (
anti-VEGF neutralizing antibody A4.6.1 had no effect on
proliferation of DU-145 in vitro (data not shown). DU-
145.luciferase, a subclone which stably expresses high levels
of luciferase activity, was generated by transfection with the (
luciferase expression vector pRK7.luciferase, with cells ex- 22 29 35 42
pressing 0.0015 RLFU/cell. As few as 10 cells can be detected
in organ lysate of 107-108cells. Moreover, luciferase activity DAYS
varies linearly with cell number over a range of 10’ to lo6 FIG. 1. Inhibition of primary tumor growth by systemic adminis-
cells. tration of monoclonal anti-VEGF antibody, A4.6.1. Mice were inoc-
SCID mi= i n d M subcutaneously with DU-145.luciferase ulated with 2 X lo6 tumor cellslanimal. Anti-VEGF therepy with
t m o r cells invariably develop measurable primary tumors as A4.6,l dosed at either 100 pg. or 10 M.,i.p., bi-weekly w a s mitiated
w i t h 7 days of DU-145.luciferase in,oc+ation. Tumor size was
well as metastasis to lungs, as detected by recovery of luciferase measured weekly. Tumor growth was smficantly inhibited m the
activity from whole organ lysates. Of note, other major organ 100 Wg.-dose group versus controls (n = 8 mice per group), (p =
sites, for example liver, do not show appreciable luciferase 0.0017, unpaired t test).
 INHIBITION OF VEGF SUPPRESSES PROSTATE CANCER PROGRESSION

3
3000 +
2.5 2500

2 2000
n
E
E
W
!
rn 1500
1.5 K
0
3c
1000

-
1

500
.5

0 Y Y Y Y Y
10 mcg 100 mcg
LUNG LUCIFERASE ACTIVITY
control
Z s s s s s
a, 0, a, a, a,

FIG. 3. Delayed anti-VEGF treatment inhibits primary prostatic

FIG. 2. Early anti-VEGF treatment suppresses lung micrometas- tumor growth. Mice were inoculated with 7.5 X lo6 tumor cells/
tasis. Luciferase activity was assayed at day 43 for all three groups. animal. Anti-VEGF antibody treatment was delayed until 4 post
While controls showed significant luciferase activity over back- tumor inoculation, utilizing 100 pg., i.p., bi-weekly. Inhibition of
ground levels, neither anti-VEGF treated group (A4.6.1 dosed at tumor growth was significant despite treatment delay (n = 8 mice
either 100 pg. or 10 Fg., i.p., bi-weekly) showed evidence of metas- per group), (p = 0.011,unpaired t test).
tasis (Fisher's exact p-value = 0.0013).

tumor cell inoculation. Despite the delay in anti-VEGF ther-

apy, further primary tumor growth was inhibited (fig. 3).
After 9 weeks, the average tumor size measured 540 2 147
mm.3 in the delayed treatment group versus 1953 2 432
mm.3 in untreated controls, (p = 0.011, unpaired t test).
Untreated, tumor-bearing mice demonstrate progressive
micro-metastatic disease over time. Control cohorts of un-
treated mice sacrificed 4, 6 and 9 weeks after tumor cell
inoculation had a progressive increase in metastatic activity
with lung luciferase activity of 1.17, 5.43, and 105.8 RLFU
measured at the 4, 6, and 9 weeks time points, respectively.
In marked contrast, when treatment was initiated at 4
weeks, there was no increase in metastatic activity over the
subsequent treatment period (lung luciferase activity = 1.21
RLFU at 9 weeks versus 1.17 RLFU at onset of treatment),
while activity in the untreated control group progressed to
high levels, measuring 105.76 RLFU at 9 weeks, as noted
above (fig. 4). Thus, despite delaying initiation of treatment
until after primary tumors were well-established and micro-
metastases had occurred, treatment with A4.6.1 again ef-
fected marked inhibition of metastatic progression (p= FIG. 4. Delayed anti-VEGF treatment suppresses lung microme-
0.0065, Mann-Whitney U test). tastasis. Untreated mice bearing tumors were examined a t 4, 6, and
9 weeks for presence of metastasis. Significant luciferase activity in
lung was found a t 4 weeks, and progressive increase in activity was
DISCUSSION noted a t 6 and 9 weeks. Concurrently, a group of mice was random-
VEGF expression, with concomitant up regulation of its ized to start anti-VEGF treatment at 4 weeks post- tumor cell injec-
tion. Treated animals were sacrificed a t 9 weeks. Initiation of ther-
receptors, KDR and flt, is increased in tumor capillaries, apy a t 4 weeks inhibited further progression of metastasis versus
suggesting a central role in tumor neovaseularization.", l 6 controls (p = 0.0065, Mann-Whitney U test).
Indeed, inhibition of VEGF by treatment with a neutralizing
antibody, A4.6.1 is sufficient to inhibit primary tumor growth
of various cancer cell line ~enografts.~.'Neutralization of spread in our model clearly differs from disseminated pros-
VEGF also inhibits various stages of metastatic progression, tate cancer in humans, the current experiments suggest that
that is both initial microscopic dissemination and subsequent anti-VEGF therapy may prove clinically useful in the setting
macroscopic outgr~wth.".~ While the pattern of metastatic of already disseminated but still micro-metastatic disease as
 INHIBITION OF VEGF SUPPRESSES PROSTATE CANCER PROGRESSION 963
well. Our pre-clinical d a t a suggest that a survey of prostate tion of the increase in vascular permeability induced by vas-
tumor specimens for VEGF expression to determine the po- cular permeability factor in vivo. Br. J. Pharmacol., 109: 195,
tential clinical relevance of such therapy may be warranted. 1993.
Indeed, i n preliminary studies, we have detected VEGF in 3. Ferrara, N.. Houck, K., Jakeman, L. and h u n g , D. W.: Molecu-
t h e urine of 11 of 18 patients with advanced prostate cancer lar and biological properties of the vascular endothelial growth
(unpublished observation). factor family of proteins. Endocrine Rev., 1 3 18,1992.
4. Brown, L. F., Berse, B. and Jackman, R. W.: Expression of
Systemic anti-angiogenic therapy can markedly inhibit tu-
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a n increased rate of apoptosis i n primary tumor cells and colon cancer tumorigenesis in a mouse model of experimental
maintains micrometastasis i n a state of dormancy by the liver metastasis. J. Clin. Invest., 95 1789,1995.
same mechanism.18 Our studies examined the effect of a 6. Boocock, C. A., Charnock-Jones, D. S. and Sharkey, A. M.: Ex-
specific anti-VEGF antibody, A4.6.1, on established prostatic pression of vascular endothelial growth factor and its recep-
carcinoma grafts already metastatic to t h e lung. By neutral- tors flt and KDR in ovarian carcinoma. JNCI, 87: 506,1995.
7. Berkman, R. A,, Merrill, M. J. and Reinhold, W. C.: Expression
izing tumor-secreted VEGF, both primary tumor growth and of the vascular permeability factor/vascular endothelial
further metastatic progression i n the lung was inhibited, growth factor gene in central nervous system neoplasms.
presumably by the arrest of further vascular recruitment by J. Clin. Invest., 91:153,1993.
both tumor primary and micrometastasis, as well as by in- 8. Kim, J. K.,Li, B. and Winer, J.: Inhibition of vascular endothe-
hibition of secondary metastasis from established metastatic lial growth factor-induced angiogenesis suppresses tumor
foci. growth in vivo. Nature, 362 841,1993.
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antibody treatment despite repeated, chronic dosing: there growth factor promotes tumor dissemination by a mechanism
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an anti-VEGF agent might inhibit physiologic processes biologically relevant regions by neutralizing monoclonal anti-
characterized by active vascular proliferation, for example, bodies. Growth Factors, 7: 153,1992.
wound healing and o v u l a t i ~ n . 'Chronic
~ ~ ~ ~ treatment with 12. Stone, K. R., Mickey, D. D., Wunderli. H., Mickey, G. H. and
an anti-VEGF agent may disrupt homeostasis within quies- Paulson, D. F.: Isolation of a human prostate carcinoma cell
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glomerulus. l 3 Current Phase I clinical trials addressing confers a growth advantage in vivo to Chinese hamster ovary
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14. Crowley, C. W., Cohen, R. L., Lucas, B. K., Liu, G., Shuman,
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all cancer deaths in men.21 Angiogenesis is an important vascular permeability factor (vascular endothelial growth fac-
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Robert L. Cohen (Genentech, Inc.) for provision of pRK517 tumours to the ovarian follicle. Int. J. Exp. Pathol., 78: 57,
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20. Peters, K.G., De Vries, C. and Williams, L. T.: Vascular endo-
thelial growth factor receptor expression during embryogene-
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