Methods of expressing angiostatic protein

Methods of expressing angiostatic protein

US005885795A
United States Patent [19]
[11]
Patent Number:
O’Reilly (it al.
[45]
Date of Patent:
5,885,795
*Mar. 23, 1999
[54]
METHODS OF EXPRESSING ANGIOSTATIC
PROTEIN
Folkman J, et al., Tumor behavior in isolated perfused
organs: In vitro groWth and metastasis of biopsy material in
rabbit thyroid and canine intestinal segments. Annals of
[75]
Inventors: Michael S. O’Reilly, Winchester; M.
Judah Folkman, Brookline, both of
Mass.; Kim Lee Sim, Gaitherburg, Md.
Surgery 164:491—502, 1966.
Gimbrone, M.A., Jr. et al., Tumor groWth and neovascular
iZation: An experimental model using the rabbit cornea. J.
Natl. Cancer Institute 52:41—427, 1974.
[73]
Assignee: The Children’s Medical Center
Corporation, Boston, Mass.
Gimbrone MA Jr., et al., Tumor dormancy in vivo by
prevention of neovasculariZation. J. Exp. Med.
[*]
Notice:
Knighton D., Avascular and vascular phases of tumor
groWth in the chick embryo. British J. Cancer, 35:347—356,
136:261—276.
The term of this patent shall not extend
beyond the expiration date of Pat. No.
5,792,845.
1977.
Lien W., et al., The blood supply of experimental liver
metastases. II. Amicrocirculatory study of normal and tumor
vessels of the liver With the use of perfused silicone rubber.
[21] Appl. No.: 429,743
[22] Filed:
Apr. 26, 1995
Related US. Application Data
[63]
Continuation-in-part of Ser. No. 326,785, Oct. 20, 1994,
which is a continuation-in-part of Ser. No. 248,629, Apr. 26,
1994, Pat. No. 5,639,725.
[51]
Int. Cl.6 .......................... .. C12N 15/00; C12P 21/06;
[52]
US. Cl. .................. .. 435/691; 435/696; 435/172.3;
[58]
Field of Search ............................ .. 424/94.63, 93.21;
C07K 14/00; C07H 21/04
530/300; 530/350; 536/231
435/212, 226, 69.1, 172.3, 320.1; 530/350,
380, 828; 536/232, 23.5; 514/44
Surgery 68:334—340, 1970.
Folkman J, et al., Induction of angiogenesis during the
transition from hyperplasia to neoplasia. Nature 339:58—61,
1989.
Kim K J, et al., Inhibition of vascular endothelial groWth
factor—induced angiogenesis suppresses tumor groWth in
vivo. Nature 362:841—844, 1993.
Hori A, et al., Suppression of solid tumor groWth by immu
noneutraliZing monoclonal antibody against human basic
?broblast groWth factor. Cancer Research, 51:6180—6184,
1991.
Gross JL, et al. Modulation of solid tumor groWth in vivo by
bFGF. Proc. Amer Assoc. Canc. Res. 31:79, 1990.
Ingber D, et al., Angioinhibins: Synthetic analogues of
fumagillin Which inhibit angiogenesis and suppress tumor
[56]
References Cited
FOREIGN PATENT DOCUMENTS
91/09118
91/10424
WO91/10424
6/1991
7/1991
7/1991
WIPO .
WIPO .
WIPO .
OTHER PUBLICATIONS
DerWent Publications, Ltd., London; JP 58—036—391 (San
kyo KK) Mar. 3, 1983 (Abstract).
M. O’Reilly et al., “The Suppression of Tumor Metastases
by a Primary Tumor” Science vol. 44, pp. 474—476; 1993.
T. E. Maione et al., “Inhibition of Angiogenesis by Recom
binant Human Platelet Factor—4 and Related Peptides” Sci
ence vol. 247, pp. 77—79; Jan. 5,1990.
M. A. Moses et al., “Identi?cation of an Inhibitor of Neovas
groWth. Nature, 48:555—557, 1990.
Weidner N, et al., Tumor angiogenesis and metastasis—
correlation in invasive breast carcinoma. N. Engl. J. Med.
324:1—8, 1991.
Weidner N, et al., Tumor angiogenesis: A neW signi?cant
and independent prognostic indicator in early—stage breast
carcinoma, J Natl. Cancer Inst. 84:1875—1887, 1992.
Weidner N, Carroll PR, Flax J, Blumenfeld W, Folkman J.
Tumor angiogenesis correlates With metastasis in invasive
prostate carcinoma. American Journal of Pathology,
143(2):401—409, 1993.
Srivastava A, et al., The prognostic signi?cance of tumor
vascularity in intermediate thickness (0.76—4.0 mm thick)
skin melanoma. Amer J. Pathol. 133:419—423, 1988.
(List continued on next page.)
culariZation from Cartilage” Science vol. 248, pp.
1408—1410; Jun. 15, 1990.
Primary Examiner—SuZanne E. Ziska
Attorney, Agent, or Firm—Jones & AskeW
Sottrup—Jensen et al., Progress in Chemical Fibrinolysis and
Thrombolysis, 3:191—209, 1978.
Degen et al., Genomics, 8:49—61, 1990.
Tomlinson et al., Journal of Biological Chemistry,
264(10):5957—5965, 1989.
[57]
ABSTRACT
The present invention comprises an endothelial inhibitor and
method of use therefor. The endothelial inhibitor is a protein
isolated from the blood or urine that is eluted as a single peak
from C4-reverse phase high performance liquid chromatog
Fidler et al., Cell, 79:185—188, Oct. 21, 1994.
Forsgren et al., FEBS Letters, 213(2):254—260, Mar. 1987.
MalinoWski et al., Biochemistry, 23:4243—4250, 1984.
Folkman J ., Tumor angiogenes: Therapeutic implications,
N. Engl. Jour. Med. 285:1182 1186, 1971.
Algire GH, et al. Vascular reactions of normal and malignant
plasminogen fragment beginning at amino acid number 98
tumors in vivo. I. Vascular reactions of mice to Wounds and
of a murine plasminogen molecule.
raphy. The endothelial inhibitor is a molecule comprising a
protein having a molecular Weight of betWeen approxi
mately 38 kilodaltons and 45 kilodaltons as determined by
reducing polyacrylamide gel electrophoresis and having an
amino acid sequence substantially similar to that of a murine
to normal and neoplastic transplants. J. Natl. Cancer Inst.
6:73—85, 1945.
20 Claims, 25 Drawing Sheets
5,885,795
Page 2
OTHER PUBLICATIONS
Nguyen M, et al., Elevated levels of an angiogenic peptide,
basic ?broblast growth factor, in urine of bladder cancer
patients. J. Natl. Cancer Inst. 85:241—242, 1993.
Folkman J ., Angiogenesis and its inhibitors in “Important
Advances in Oncology 1985 ”, VT DeVita, S. Hellman and
S. Rosenberg, editors, J.B. Lippincott, Philadelphia 1985.
Obeso, et al., “Methods in Laboratory Investigation, A
Robbins, K.C., “The plasminogen—plasmin enzyme system”
Hemangioendothelioma—derived cell line; Its use as a Model
Hemostasis and Thrombosis, Basic Principles and Practice,
2nd Edition, ed. by Colman, R.W. et al. J.B. Lippincott
Company, pp. 340—357, 1987.
for the Study of Endothelial Cell Biology,” Lab Invest.,
63(2), pp. 259—269, 1990.
Yoshimura, T, et al., “Cloning, sequencing, and expression
of human macrophage stimulating protein (MSP, MST1)
Sotrrup—Jensen, L., et al., Progress in Chemical Fibrinolysis
and Thrombolysis, vol. 3, Davidson, J .F., et al. eds. Raven
con?rms MSP as a member of the family of kringle proteins
Press, NeW York, p. 191 (1978).
and locates the MSP gene on Chromosome 3” J. Biol.
Grant, D.S., et al., “Scatter factor induces blood vessel
formation in vivo,” Proc. Natl. Acad. Sci, vol. 90, pp.
Chem.., vol. 268, No. 21, pp. 15461—15468, 1993.
BroWne, M.J., et al., “Expression of recombinant human
plasminogen and aglycoplasminogen in HeLa cells” Fibrin
olysis vol. 5
257—260, 1991.
Brem et al., “Interstitial chemotherapy With drug polymer
implants for treatment of recurrent gliomas,” J. Neurosurg.
74:441—446 (1991).
Muthukkaruppan Vr., et al., Angiogenesis in the mouse
cornea. Science 205:1416—1418, 1979.
Passaniti A, et al., “A simple quantitative method for assess
ing angiogenesis and anti—angiogenic agents using recon
stituted basement membrane, heparin and ?broblast groWth
factor,” Lab. Invest., vol. 67, p. 519 (1992).
1937—1941 (1993).
Shi, G. et al., “Kringle Domains and Plasmin Denaturation,”
Biochem. and Biophys. Res. Comm., vol. 178, No. 1, pp.
360—368 (1991).
Scaller, J. et al., “Structural Aspects of the Plasminogen of
Various Species,” Enzyme, vol. 40, pp. 63—69 (1988).
Wiman, B., et al., “On the Speci?c Interaction betWeen the
Lysine—binding sites in Plasmin and Complementary Sites
in <<2—Antiplasmin and in Fibrinogen,” Biochemica et Bio
physica Acta, 579, 142 (1979).
U.S. Patent
Mar. 23, 1999
Sheet 1 0f25
5,885,795
Mouse Plasminogen Sequence:
met
pro
ala
val
asp
gly
ser
ser
his
gln
leu
asp
lys
gly
phe
cys
glu
asp
ser
leu
val
ser
leu
ala
cys arg ser phe gln tyr
phe
tyr
gln
gly
his ser lys glu
met ala glu asn ser
thr ser ser ile ile arg met arg asp
lys
ile
leu
thr
lys
leu
asp
lys
cys
leu
gly
lys
glll
leu
ile
leu
glu
leu
ser
ala
thr
leu
thr
ala
asp
leu
gln
gly
phe
lys
gly
gly
val
gln gln cys val ile
val ile leu phe glu lys arg val tyr leu ser glu cys lys thr
gly ile gly asn gly tyr arg gly thr met ser arg thr lys ser
gly val ala cys gln lys trp gly ala thr phe pro his val pro
aSn tyr ser pro ser thr his pro ash glu gly leu glu glu asn
tyr cys arg ash pro asp asn asp glu gln gly pro trp cys _tyr
thr thr asp pro asp lys arg tyr asp tyr cys ash ile pro glu
cys glu glu glu cys met tyr cys ser gly glu lys tyr glu gly
lys ile ser lys thr met ser gly leu asp cys gln ala trp asp
ser gln ser pro his ala his gly tyr ile pro ala lys phe pro
ser
lys
ash
leu
lys met
asn tyr cys his ash pro asp gly glu
pro arg pro
trp cys phe
thr thr asp pro thr lys arg trp glu
tyr cys
ile
cys
asp
pro arg
thr
thr pro pro pro pro pro
ser
pro thr tyr gln cys leu lys gly arg gly glu ash tyr arg gly
thr val ser val thr val ser gly lys thr cys gln arg trp ser
glu gln thr pro his arg his asn arg thr pro glu ash phe pro
cys lys ash leu glu glu ash tyr cys arq ash pro asp gly qlu
thr ala pro trp cys tyr thr thr asp ser gln leu arg trp giu
tyr cys glu ile pro ser cys glu ser ser ala ser pro asp gln
Ser asp ser ser val pro pro glu glu gln thr pro val val gln
glu cys tyr gln ser asp gly gln ser tyr arg gly thr ser ser
thr thr ile thr gly lys lys cys gln ser trp ala ala met phe
pro his arg his ser lys thr pro glu asn phe pro asp ala gly
leu glu' met ash tyr cys arg asn prO asp gly asp lys gly Pro
trp cys
tyr
thr
thr asp pro ser val ar
trp glu
tyr cys ash
leu lys arg cys ser glu thr gly gly ser val val glu leu pro
thr val
ser gln glu pro
ser gly pro ser asp ser glu
thr asp
cys met tyr gly asn gly lys asp tyr arg gly lys thr ala val
thr ala ala gly thr pro cys gln gly trp ala ala gln glu pro
his
leu
pro
asp
arg
glu
trp
ile
his ser ile
lys asn tyr
cys tyr thr
pro leu cys
phe thr pro gln
cys arg ash pro
thr ash pro arg
ala ser ala ser
thr
asp
lys
ser
ash
gly
leu
phe
pro
asp
tyr
glu
arg
val
asp
cys
ala
asn
tyr
gly
asp
gly
cys
lys
pro gln val glu pro lys
lys cys pro gly arg val val gly gly
Cys val
ser trp pro trp gln ile
ala
ash pro his
ser
leu arg
thr arg phe thr gly gln his phe cys gly gly thr leu ile ala
pro glu trp val
leu thr ala ala his cys leu glu lys ser ser
arg pro glu phe tyr lys val ile leu gly ala his glu glu tyr
ile arg gly leu asp val gln glu ile ser val ala lys leu ile
leu glu pro ash ash arg
pro ala thr ile thr asp
asp ile ala leu leu lys leu ser arg
lys val ile pro ala cys leu pro ser
U.S. Patent
Mar. 23, 1999
Sheet 2 0f25
5,885,795
pro asn tyr met; val ala asp arg thr ile cys tyr ile thr gly
crp gly glu thr gln gly thr phe gly ala gly arg leu lys glu
ala gln leu pro val ile glu asn lys val cys asn arg val glu
tyr leu asn asn arg val lys ser thr glu leu cys ala gly gln
leu ala gly gly val asp ser cys gln gly asp ser gly gly pro
leu val cys phe glu lys asp lys tyr ile leu gln gly val thr
ser trp gly leu gly cys ala arg pro asn lys pro gly val tyr
val arg val ser arg phe val asp trp ile glu arg glu met arg
asn asn
U.S. Patent
Mar. 23, 1999
Sheet 3 0f25
5,885,795
MOUSE
HUMAN
RHESU S MONKEY
PORC INE
BOVINE
val
val
val
ile
ile
tyr
tyr
tyr
tyr
tyr
leu
leu
leu
leu
leu
ser
ser
ser
ser
leu
glu
glu
glu
glu
glu
cys
cys
cys
cys
cys
lys
lys
lys
lys
lys
thr
thr
thr
thr
thr
gly
gly
gly
gly
gly
ile
asn
asn
asn
asn
gly
gly
gly
qly
gly
asn
lys
lys
lys
gln
gly
asn
asn
asn
thr
tyr
tyr
tyr
tyr
tyr
arg
arg
arg
arg
arg
gly
gly
gly
gly
gly
thr
thr
thr
thr
thr
met
met
met
thr
thr
ser
ser
ser
ser
ala
arg
lys
lys
lys
glu
thr
thr
thr
thr
thr
lys
lys
arg
lys
lys
ser
asn
thr
ser
ser
gly
gly
gly
gly
gly
val
ile
ile
val
val
ala
thr
thr
lle
thr
cys
cys
cys
cys
CyS
gln
gln
gln
gln
gln
lys trp
lys trp
lys.trp
lys trp
lys trp
gly
ser
ser
ser
ser
ala
ser
ser
val
ala
thr
thr
thr
ser
thr
phe
ser
ser
ser
ser
pro
pro
pro
pro
pro
his
his
his
his
his
val
arg
arg
ile
val
pro
pro
pro
pro
pro
asn
arg
thr
lys
lys
tyr
phe
phe
tyr
phe
ser
ser
ser
ser
ser
pro
pro
pro
pro
pro
ser
ala
ala
glu
glu
thr
thr
thr
lys
lys
his
his
hlS
phe
phe
asn
ser
ser
leu
leu
glu
glu
glu
ala
ala
pro
pro
pro
pro
pro
gly leu glu glu asn tyr cys arg asn pro asp asn asp glu gln gly
gly leu glu glu asn tyr cys arg asn pro asp asn asp pro gln gly
gly leu glu glu asn tyr cys arg asn pro as}? asn asp gly gln gly
gly leu glu glu asn tyr cys arg asn pro asp asn asp glu lys gly
gly leu glu glu asn tyr cys arg asn pro asp asn asp glu asn gly
pro
pro
pro
pro
pro
trp
trp
trp
trp
trp
cys
cys
cys
cys
cys
tyr
tyr
tyr
tyr
tyr
thr
thr
thr
thr
thr
thr
thr
thr
thr
thr
ile
ile
ile
ile
pro
leu
pro
pro
glu
glu
glu
glu
ile pro glu
cys
cys
cys
cys
cys
glu
glu
glu
glu
glu
glu
asp
asp
asp
asp
asp
asp
asp
glu
glu
glu
glu
glu asp lys
pro asp
pro'glu
pro glu
pro glu
pro asp
lys
lys
glu
thr
lys
arg
arg
arg
arg
arg
tyr
tyr
phe
phe
tyr
asp
asp
asp
asp
asp
tyr
tyr
tyr
tyr
tyr
cys
cys
cys
cys
cys
asn
asp
asp
asp
asp
tyr
his
his
his
cys met his
cys
cys
cys
CYS
cys
ser
Ser
ser
ser
ser
gly
gly
gly
gly
gly
glu
glu
glu
glu
glu
lys
asn
asn
his
asn
tyr
tyr
tyr
tyr
tyr
cys
cys
cys
cys
met
met
met
met
glu gly lys ile ser lys thr met ser gly leu asp cys gln ala trp
asp gly lys ile ser lys thr met ser gly leu glu cys gln ala trp
asp gly lys ile ser lys thr met ser gly ‘leu g1u cys gln ala trp
glu gly lys ile ser lys thr met ser gly 116 glu Cys gln ser trp
glu gly lys ile ala lys thr met ser gly arg asp Cys gln ala trp
U.S. Patent
Mar. 23, 1999
ser
gln
ser
pro his ala his
ser
ser
ser
ser
gln
gln
gln
gln
ser
ser
ser
ser
pro
pro
pro
pro
his
his
his
his
ala
ala
ala
ala
his
his 91y
his gly
his gly
leu
leu
leu
leu
leu
lys met
lys lys
lys lys
lys met
lys me C
asn
asn
asn
asn
ELSE
cys
cys
cys
cys
asn
lys
lys
lys
lys
lys
arg
arg
arg
arg
arg
pro
pro
pro
pro
pro
cys
cys
cys
cys
cys
phe
phe
phe
phe
phe
asp
asp
asp
asp
asp
ile
ile
ile
asp
ile pro ala lys phe pro
ser lys phe pro
ser lys phe pro
leu pro ser lys phe pro
ile pro ser lys phe pro
gly
asp
asp
asp
91y
5,885,795
Sheet 4 0f 25
ile pro
ile pro
asp gly
asp arg
asp gly
asp gly
asp gly
glu pro
glu leu
glu pro
glu pro
glu pro
cys
asn
asn
asn
asn
asn
pro
pro
pro
pro
pro
thr
thr
thr
thr
thr
pro
pro
pro
pro
pro
lys
lys
lys
lys
lys
arg
arg
arg
arg
arg
cys thr thr
cys thr thr
cys thr thr
cys thr thr
cys thr thr
pro
pro
pro
pro
pro
pro pro
ser
pro thr
ser
ser
ile
ile
arg
arg
arg
arg
arg
gly
sly
qly
sly
pro thr tyr
pro thr tyr
pro thr tyr
pro lys tyr
gln
gln
gln
gln
gln
cys
cys
cys
cys
cys
lys
lys
lys
lys
lys
gly arg
gly thr
gly thr
gly arg
<arly thr
91y
gly
gly
gly glu
gly
asn
asn
asn
arg
arg
arg
EiSI'l
arg
asn
gly
thr
thr
thr
thr
thr
val
ser
ser
ala
ser
S81”
his
his
his
his
thr
thr
thr
thr
arg
cys
his
cys
cys his 91y
‘ arg
cys
glu
qly
sly
gly
qly
gly
lys thr
val
val
cys
arg
arg
arg
arg
arg
thr
thr
thr
thr
thr
pro
pro
glu
glu
pro
pro
pro
glu
glu
arg
arg
asn
asn
asn
asn
asn
pro
pro
pro
pro
pro
asp
asp
asp
asp
asp
ser
asn
asn
asn
arg his
thr his
thr his
lys his
lys his
asn
asn
asn
asn
asn
tyr
tyr
SE1"
asn
asn
asn
asn
asn
cys
cys
tyr cys
tyr cys
tyr cys
arg
arg
arg
thr
thr
thr
thr
thr
glu
thr
ser
ser
S81’
ser
ser
ser
ser
-
glu tyr cys
olu le’u cys
glu leu cys
glu phe cys
glu phe cys
qly thr val
qly
gly
gly
asn
ser
tyr
val
va 1 ala val
asp val ala val
thr val ser val
thr va 1 ala val
glu gln
ala gln
ala gln
ala gln
thr pro his
thr pro his
thr pro his
ser
ser
ser
glu gln thr pro his
asn
phe
phe
phe
phe
phe
cys
cys
cys
cys
cys
lys
lys
lys
lys
lys
gly
gly
qly
qly
gly
glu thr ala
lys arg ala
glu lys ala
glu thr ala
glu lys ala
pro
pro
pro
pro
pro
arg
asn
asn
asn
asn
pro
pro
pro
pro
pro
2B
asn
asn
asn
asn
asn
ser
pro his
leu glu glu
leu asp glu
leu asp glu
leu glu glu
leu glu glu
cys tyr thr
cys his thr
cys tyr thr
cys tyr thr
cys tyr thr
U.S. Patent
Mar. 23, 1999
thr asp
thr asn
thr asn
thr asp
thr asn
ser
ser
ser
ser
ser
gln
gln
gln
glu
glu
leu
val
val
val
ser
ser
ser
ser
ser
ala
pro
pro
thr
pro
ser
pro asp
val
val
thr
leu
ser
ser
ser
ser
ser
ser
glu tyr
glu tyr
glu tyr
arg
arg
arg
arg
val arg
ser
ser
ser
asp tyr
glu tyr
ser
pro val val gln
leu thr pro val val gln
leu thr pro val val gln
gln thr pro val ala gln
arg
arg
arg
arg
arg
gly
gly
gly
gly
gly
pro val pro
gln
thr
thr
thr
thr
thr
thr
thr
thr
thr
thr
ile thr
thr thr
thr thr
ile thr
ile thr
ala ala met
ser
ser
val
ser
gly
gly
gly
ser
ser
ser
ser
ser
ser
ser
SE11‘
ser
ser
phe pro his
thr
thr
thr
thr
pro
pro
pro
pro
his
his
his
his
gly
gly
gly
gly
ala gly
leu
leu
leu
leu
leu
glu
met
met
met
met
met
asp ala
ala
ala
ala
asn
asn
asn
asn
ser
met
met
met
met
ser
ser
ser
ser
pro
pro
pro
pro
pro
tyr
tyr
tyr
tyr
tyr
cys
cys
cys
cys
cys
ser
thr
thr
thr
thr
ile
ile
ile
ile
ile
pro
pro
pro
pro
pro
ser
cys
cys
cys
cys
cys
ser
val pro
thr ala
thr ala
pro val
pro val
pro
pro
pro
pro
pro
pro
pro
pro
pro
ser
pro
pro leu asp pro
his leu asp ala
arg met asp val
gln thr
gln thr
asp
cys glu
cys lys
cys lys
cys lys
cys thr
gln leu ala
thr
thr
thr
thr
asn
asn
asn
asn
asn
5,885,795
Sheet 5 0f 25
gln
ser
ser
ser
ser
glu
gly gln
ser
his gly asp gly gln
his gly asp gly gln
ser
gly
his gly
ser
ser
ser
asp
gly glu
gly gln
arg
asn
asn
gly
gly
gly
gly
gly
lys lys cys
lys lys cys
lys lys cys
arg lys cys
arg lys cys
his
his
his
his
his
ser lys thr
gln lys thr
glu lys thr
glu lys thr
leu lys thr
pro
pro
pro
pro
pro
glu
glu
gly
glu
tyr
tyr
tyr
tyr
tyr
cys
cys
cys
cys
cys
pro
pro
pro
pro
pro
asp
asp
asp
asp
asp
arg
arg
arg
arg
arg
asn
asn
asn
asn
asn
glu
ser
gln
gln
gln
gln
gln
SEI~
asn
asn
asn
asn
asn
phe pro
tyr pro
phe pro
phe pro
tyr pro
ser
ser
ser
ser
gly asp lys
ala asp lys
ala asp
ala asp
ala asp
lys
lys
lys
U.S. Patent
Mar. 23, 1999
Sheet 6 0f25
5,885,795
O
ZSOQ~Hh<MR-i.Um7=
mw oM_H~{5rm Em
TA
um OCRT
w
mm
.
mm
_
F
ID
3TR AME
DAYS AFTER OPERATION
0.8*
avEO2E5?
6
4.
.
0.
O
2
0.0
IN
|O
I INTACT TUMOR
[I] TUMOR RESECTED
l5
DAYS AFTER OPERATION
3.0 -
:zio2qu9mz
I NORMAL MICE
[I TUMOR BEARING MICE
0.0
2. 5 ng/ml
50 ng/ml
U.S. Patent
'1 UMOR
INTACT
$5EQS25? 2?5E9Hu3cm?.-5
Mar. 23, 1999
Sheet 9 0f25
5,885,795
TUMOR REMOVED
@SZumH-K0DA AE<I2SMODZ
TUMOR
‘__— TUMOR REMOVED ‘—_——.
INTACT
U.S. Patent
Mar. 23, 1999
Sheet 10 0f25
5,885,795
U.S. Patent
Mar. 23, 1999
Sheet 11 0f25
5,885,795
U.S. Patent
Mar. 23, 1999
Sheet 12 0f25
5,885,795
BOVINE
CAPILLARY ENDOTHELIAL CELL ASSAY OF RECOMBINANT HUMAN ANGIOSTATIN
—D—-
A] RECOMBINANT HAS
......
A2 RECOMBINANT HAS
1OO_
BlRECOMBlNANT HAS
6}
‘\\\
‘\
9..
75 _
\
\_\
\\
?
25—
15‘
B2 RECOMBINANT HAS
- - -EE- - -
C1 CONTROL
- - - - o- - --
c2 CONTROL
———$-- -
D1 CONTROL
- - V- —
D2 CONTROL
- --B-- —
E RECOMBINANT HAS
\
\\
%INHBTO
----A----
1:10
Dilution
U.S. Patent
Mar. 23, 1999
Sheet 13 0f25
5,885,795
BOVINE CAPILLARY ENDOTHELIAL CELL ASSAY OF RECOMBINANT HUMAN ANGIOSTATIN
T
_
_
_
20Q5 “27:
TT
N
T
Em
EEK MMM HumMZMS
Mmm
m
u
?
m OACME/506
RRR
A5
MmqozEeIQun
%
w
w
m
2o
0
DILUTION
_13
U.S. Patent
Mar. 23, 1999
Sheet 14 0f25
5,885,795
U.S. Patent
A35$2:3;
O0
Mar. 23, 1999
Sheet 15 0f25
5,885,795
,v.
4.
a. _
FJSA
QZ:zE5;Q
Q52 :;9.2
U.S. Patent
Mar. 23, 1999
Sheet 16 0f25
5,885,795
000
0.0 f I
O
068
9
DAYS
I3
I
_ 16A
IOO
_
0
2
_
_
_
_
O6
8420
gX0H:Q6;2Z0~:
“if
F_ 16B
m!
12
_ 16C
U.S. Patent
Mar. 23, 1999
Sheet 17 0f25
5,885,795
TREATMENT OF T241 PRIMARY TUMORS WITH ANGIOSTATIN
(40 mg/kg)
100005
S ALm
B000 -
A@n2Q3OE1!DV’H
6000 -
4000 -
AN GIOSTATIN
2000 -
TREATMENT DAY
17
TREATMENT OF LLC-LM PRIMARY TUMORS WITH ANGIOSTATIN
(80 mg/kg/day)
|2000—
SALINE
$@~2539»?
B000
4000 -
ANGIOSTATIN
TREATMENT DAY
U.S. Patent
Mar. 23, 1999
Sheet 18 0f 25
12000 T241 Fibrosarcoma
5,885,795
Saiine
&
“EE
8000
\o
O)
E
2
O
>
8
E 4w)
=
+3
Angiostatin
l
0
5
\o
l
15
2o
Treatment Day
FIGURE 19
25